Translation control elements for high-level protein expression in the plastids of higher plants and methods of use thereof

ABSTRACT

DNA constructs containing translational control elements are provided. These 5′ regulatory segments facilitate high level expression of transgenes introduced into the plastids of higher plants.

This application is a divisional application of U.S. patent applicationSer. No. 09/762,105 now U.S. Pat. No. 6,987,215 filed Apr. 23, 2001which claims priority to PCT/US99/17806, filed Aug. 3, 1999, U.S.Provisional Application 60/095,163, 60/112,257, 60/095,167, 60/131,611and 60/138,764. The entire disclosure of each of the foregoingapplications is incorporated by reference herein.

Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Science Foundation, GrantNumber MCB-96-30763.

FIELD OF THE INVENTION

This invention relates to the fields of transgenic plants and molecularbiology. More specifically, the invention provides vectors targeting theplastid genome which contain translation control elements facilitatinghigh levels of protein expression in the plastids of higher plants. Bothmonocots and dicots are successfully transformed with the DNA constructsprovided herein.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application in order to morefully describe the state of the art to which this invention pertains.The disclosure of each of these publications is incorporated byreference herein.

The chloroplasts of higher plants accumulate individual components ofthe photosynthetic machinery as a relatively large fraction of totalcellular protein. The best example is the enzymeribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) involved inCO₂ fixation which can make up 65% of the total leaf protein (Ellis, R.J. 1979). Because of the potentially attainable high protein levels,there is significant interest in exploring chloroplasts as analternative system for protein expression. To date, protein levelsexpressed from transgenes in chloroplasts are below the levels ofhighly-expressed chloroplast genes. Highest levels reported thus far inleaves are as follows: 1% of neomycin phophotransferase (Carrer et al.,1993); 2.5% β-glucuronidase (Staub and Maliga, 1993) and 3-5% ofBacillus thuringiensis (Bt) crystal toxins (McBride et al., 1995). Analternative system, based on a nuclear-encoded, plastid-targeted T7 RNApolymerase may offer higher levels of protein expression (McBride t al.,1994), although this yield may come at a price.

In bacteria, the rate limiting step of protein synthesis is usually theinitiation of translation, involving the binding of the initiator tRNA(formyl-methionyl-tRNA_(f)) and mRNA to the 70S ribosome, recognition ofthe initiator codon, and the precise phasing of the reading frame of themRNA. Translation initiation depends on three initiation factors (IF1,IF2, IF3) and requires GTP. The 30S subunit is guided to the initiationcodon by RNA-RNA base pairing between the 3′ of the 16S rRNA and themRNA ribosome binding site, or Shine-Dalgarno (SD) sequence, locatedabout 10 nucleotides upstream of the translation initiation codon(Voorma, 1996). RNA-RNA interaction between the “downstream box” (DB), a15 nt sequence downstream of the AUG translational initiation codon andcomplementary sequences in the 16S rRNA 3′ sequence or anti-downstreambox (ADB; nucleotide positions 1469-1483) may also facilitate loading ofthe mRNA onto the 30S ribosome subunit (Sprengart et al., 1996). Inaddition, specific protein-RNA interactions may also facilitatetranslation initiation (Voorma, 1996).

Key components of the prokaryotic translation machinery have beenidentified in plastids, including homologues of the bacterial IF1, IF2and IF3 initiation factors and an S1-like ribosomal protein (Stern etal., 1997). Most plastid mRNAs (92%) contain a ribosome binding site orSD sequence: GGAGG, or its truncated tri- or tetranucleotide variant.This sequence is similar to the bacterial SD consensus 5′-UAAGGAGGUGA-3′(SEQ ID NO: 28; Voorma, 1996). High level expression of foreign genes ofinterest in the plastids of higher plants is extremely desirable. Thepresent invention provides novel genetic translational control elementsfor use in plastid transformation vectors. Incorporation of theseelements into such vectors results in protein expression levelscomparable to those observed for highly expressed chloroplast genes inboth monocots and dicots.

SUMMARY OF THE INVENTION

5′ genetic regulatory regions contain promoters with distinct DNAsequence information which facilitates recognition by the RNA polymeraseand translational control elements which facilitate translation. Both ofthese components act together to drive gene expression.

In accordance with the present invention, chimeric 5′ regulatory regionshave been constructed which incorporate translation control elements.Incorporation of these chimeric 5′ regulatory regions into plastidtransforming vectors followed by transformation of target plant cellsgives rise to dramatically enhanced levels of protein expression. Thesechimeric 5′ regulatory regions may be used to advantage to expressforeign genes of interest in a wide range of plant tissues. It is anobject of the present invention to provide DNA constructs and methodsfor stably transforming plastids of multicellular plants containing suchpromoters.

In one embodiment of the invention recombinant DNA constructs forexpressing at least one heterologous protein in the plastids of higherplants are provided. The constructs comprise a 5′ regulatory regionwhich includes a promoter element, a leader sequence and a downstreambox element operably linked to a coding region of said at least oneheterologous protein. The chimeric regulatory region acts to enhancetranslational efficiency of an mRNA molecule encoded by said DNAconstruct. Vectors comprising the DNA constructs are also contemplatedin the present invention. Exemplary DNA constructs of the inventioninclude the following chimeric regulatory regions: PrnnLatpB+DBwt,PrrnLatpB−DB, PrrnLatpB+DBm, PrrnLclpP+DBwt, PrrnclpP−DB,PrrnLrbcL+DBwt, PrrnLrbcL−DB, PrrnLrbcL+DBm, PrrnLpsbB+DBwt,PrrnLpsbB−DB, PrrnLpsbA+DBwt, PrrnLpsbA−DB, PrrnLpsbA−DB(+GC),PrrnLT7g10+DB/Ec, PrrnLT7g10+DB/pt, and PrrnLT7g10−DB. Downstream boxsequences preferred for use in the constructs of the invention have thefollowing sequences: 5′ TCCAGTCACTAGCCCTGCCTTCGGCA ′3 (SEQ ID NO:29) and5′ CCCAGTCATGAATCACAAAGTGGTAA ′3. (SEQ ID NO:30)

The 5′ regulatory segments of the invention have been successfullyemployed to drive the expression of the bar gene from S. hydroscopicusin the plastids of higher plants. Synthetic bar genes have also beengenerated and expressed using the DNA constructs of the presentinvention. These constructs have been engineered to maximize transgenecontainment in plastids by incorporating rare codons into the codingregion that are not preferred for protein translation in microorganismsand fungi.

In yet another embodiment of the invention, at least one fusion proteinis produced utilizing the DNA constructs of the invention. An exemplaryfusion protein has a first and second coding region operably linked tothe 5′ regulatory regions described herein such that production of saidfusion protein is regulated by said 5′ regulatory region. In oneembodiment the first coding region encodes a selectable marker gene andthe second coding region encodes a fluorescent molecule to facilitatevisualization of transformed plant cells. Vectors comprising a DNAconstruct encoding such a fusion protein are also within the scope ofthe present invention. An exemplary fusion protein consists an aadAcoding region operably linked to a green fluorescent protein codingregion. These moieties may be linked by peptide linkers such asELVEGKLELVEGLKVA (SEQ ID NO: 104) and ELAVEGKLEVA (SEQ ID NO: 105).

Plasmids for transforming the plastids of higher plants, are alsoincluded in the present invention. Exemplary plasmids are selected fromthe group consisting of pHK30(B), pHK31(B), pHK60, pHK32(B), pHK33(B),pHK34(A), pHK35(A), pHK64(A), pHK36(A), pHK37(A), pHK38(A), pHK39(A),pHK40(A), pHK41(A), pHK42(A), pHK43(A), pMSK56, pMSK57, pMSK48, pMSK49,pMSK35, pMSK53 and pMSK54.

Transgenic plants, both monocots and dicots harboring the plasmids setforth above are also contemplated to be within the scope of theinvention.

In yet another embodiment of the invention, methods are provided forproducing transplastomic monocots. One method comprises a) obtainingembryogenic cells; b) exposing said cells to a heterologous DNA moleculeunder conditions whereby said DNA enters the plastids of said cells,said heterologous DNA molecule encoding at least one exogenous protein,said at least one exogenous protein encoding a selectable marker; c)applying a selection agent to said cells to facilitate sorting ofuntransformed plastids from transformed plastids, said cells containingtransformed plastids surviving and dividing in the presence of saidselection agent; d) transferring said surviving cells to selective mediato promote plant regeneration and shoot growth; and e) rooting saidshoots, thereby producing transplastomic monocot plants. Theheterologous DNA molecule may be introduced into the plant cell via aprocess selected from the group consisting of biolistic bombardment,Agrobacterium-mediated transformation, microinjection andelectroporation. In one embodiment of the above described method,protoplasts are obtained from the embryogenic cells and the heterologousDNA molecule is delivered to said protoplasts by exposure topolyethylene glycol. Suitable selection agents for the practice of themethods of the invention are streptomycin, and paromomycin. Monocotplants which may be transformed using the methods of the inventioninclude but are not limited to maize, millet, sorghum, sugar cane, rice,wheat, barley, oat, rye, and turf grass.

In a preferred embodiment a method for producing transplastomic riceplants is provided. This method entails the following steps: a)obtaining embryogenic calli; b) inducing proliferation of calli onmodified CIM medium; c) obtaining embryogenic cell suspensions of saidproliferating calli in liquid AA medium;

d) bombarding said embryogenic cells with microprojectiles coated withplasmid DNA;

e) tranferring said bombarded cells to selective liquid AA medium; f)transferring said cells surviving in AA medium to selective RRMregeneration medium for a time period sufficient for green shoots toappear; and

g) rooting said shoots in a selective MS salt medium.

Plasmids suitable for transforming rice as set forth above includepMSK35 and pMSK53, pMSK54 and pMSK49. Transplastomic rice plants soproduced are also contemplated to be within the scope of the invention.

In yet a final embodiment of the invention methods for containingtransgenes in transformed plants are provided. An emplary methodincludes the following steps: a) determining the codon usage in saidplant to be transformed and in microbes found in association with saidplant; and b) genetically engineering said transgene sequence via theintroduction of rare microbial codons to abrogate expression of saidtransgene in said plant associated microbe. In an exemplary embodimentof the method described immediately above the transgene is a bar geneand said rare codons are arginine encoding codons selected from thegroup consisting of AGA and AGG, and transgene is not expressed in E.coli.

The following definitions will facilitate the understanding of thesubject matter of the present invention:

Heteroplastomic: refers to the presence of a mixed population ofdifferent plastid genomes within a single plastid or in a population ofplastids contained in plant cells or tissues.

Homoplastomic: refers to a pure population of plastid genomes, eitherwithin a plastid or within a population contained in plant cells andtissues. Homoplastomic plastids, cells or tissues are genetically stablebecause they contain only one type of plastid genome. Hence, they remainhomoplastomic even after the selection pressure has been removed, andselfed progeny are also homoplastomic. For purposes of the presentinvention, heteroplastomic populations of genomes that are functionallyhomoplastomic (i.e., contain only minor populations of wild-type DNA ortransformed genomes with sequence variations) may be referred to hereinas “functionally homoplastomic” or “substantially homoplastomic.” Thesetypes of cells or tissues can be readily purified to a homoplastomicstate by continued selection.

Plastome: the genome of a plastid.

Transplastome: a transformed plastid genome.

Transformation of plastids: stable integration of transforming DNA intothe plastid genome that is transmitted to the seed progeny of plantscontaining the transformed plastids.

Selectable marker gene: the term “selectable marker gene” refers to agene that upon expression confers a selective advantage to the plastidsand a phenotype by which successfully transformed plastids or cells ortissues carrying the transformed plastid can be identified.

Transforming DNA: refers to homologous DNA, or heterologous DNA flankedby homologous DNA, which when introduced into plastids becomes part ofthe plastid genome by homologous recombination.

Operably linked: refers to two different regions or two separate genesspliced together in a construct such that both regions will function topromote gene expression and/or protein translation.

The detailed description as follows provides examples of preferredmethods for making and using the DNA constructs of the present inventionand for practicing the methods of the invention. Any molecular cloningand recombinant DNA techniques not specifically described are carriedout by standard methods, as generally set forth, for example in Sambrooket al., “DNA Cloning, A Laboratory Manual,” Cold Spring HarborLaboratory, 1989.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A. Plastid mRNAs and the small (16S) ribosomal RNA containcomplementary sequences downstream of AUG implicating interactionsbetween mRNA and 16S rRNA during translation initiation in plastids.Proposed model is based on data in E. coli (Sprengart et al., 1996); forsequence of 16S rRNA (SEQ ID NO: 108) see ref. (Shinozaki et al.,1986b). SD, Shine-Dalgarno sequence; ASD, anti SD region; DB, downstreambox; ADB, anti DB region. Watson-Crick (line) and G-U (closed circle)pairing are marked.

FIG. 1B. Sequence of the anti-downstream-box regions (ADB sequenceunderlined) of the 16S rRNA in plastids (pt; (SEQ ID NO: 109); thisapplication) and in E. coli (Ec; SEQ ID NO: 110; Sprengart et al.,1996). The E. coli ADB box contains sequences between nucleotides1469-1483 of the 16S rRNA (Sprengart et al., 1996), corresponding tonucleotides 1416-1430 of the tobacco 16S rRNA (Dams et al., 1988;sequence between nucleotides 104173-104187 in Shinozaki et al., 1986).

FIG. 2A. Base-pairing between plastid ADB (SEQ ID NO: 109) and wild typeatpB (SEQ ID NO: 111), mutant atpB (SEQ ID NO: 112), clpP (SEQ ID NO:113), wild type rbcL (SEQ ID NO: 114), mutant rbcL (SEQ ID NO: 115),psbB (SEQ ID NO: 116) and psbA (SEQ ID NO: 117) and atpB, clpP, rbcL,psbB and psbA mRNAs (underlined). Multiple alternative DB-ADBinteractions are shown. Nucleotides changed to reduce or alter mRNA-rRNAinteraction are in lower case. The number of potential nucleotide pairsformed with the 26 nt ADB region is in parenthesis. The number ofpairing events affected by mutagenesis is in bold.

FIG. 2B. Complementarity of Prrn T7 phage gene 10 leader derivatives(T7g10, SEQ ID NO: 118; T7g10+DB/Ec, SEQ ID NO: 119; T7g10+DB/pt, SEQ IDNO: 120; T7g10−DB, SEQ ID NO: 121) with the E. coli (SEQ ID NO: 110) andplastid (SEQ ID NO: 109) ADB sequences. Nucleotides changed to reduce oralter mRNA-rRNA interaction are in lower case. The number of potentialnucleotide pairs formed with the 26 nt ADB region is in parenthesis.

FIG. 3A. DNA sequence of the chimeric Prrn plastid promoter fragmentswith atpB and clpP translation control regions (PrrnLatpB+DBwt, SEQ IDNO: 1; PrrnLatpB−DB, SEQ ID NO: 2; PrrnLatpB+DBm, SEQ ID NO: 3;PrrnLclpP+DBwt, SEQ ID NO: 4; PrrnLclpP−DB, SEQ ID NO: 5). The plasmidname that is the source of the promoter fragment is given inparenthesis. The Prrn promoter sequence is underlined; nucleotide atwhich transcription initiates in tobacco plastids is marked with filledcircle; translational initiation codon (ATG) is in bold; SD isunderlined with a wavy line; nucleotides of the 5′ and 3′ restrictionsites and point mutations are in lower case.

FIG. 3B. DNA sequence of the chimeric Prrn plastid promoter fragmentswith rbcL and psbB translation control regions (PrrnLrbcL+DBwt, SEQ IDNO: 6; PrrnLrbcL−DB, SEQ ID NO: 7; PrrnLrbcL+DBm, SEQ ID NO: 8;PrrnLpsbB+DBwt, SEQ ID NO: 9; PrrnLpsbB−DB, SEQ ID NO: 10). For detailssee description of FIG. 3A.

FIG. 3C. DNA sequence of the chimeric Prrn plastid promoter fragmentswith psbA translation control regions (PrrnLpsbA+DBwt, SEQ ID NO: 11;PrrnLpsbA−DB, SEQ ID NO: 12; PrrnLpsbA−DB(+GC), SEQ ID NO: 13). Fordetails see description of FIG. 3A.

FIG. 3D. DNA sequence of the chimeric Prrn plastid promoter fragmentswith the T7 phage gene 10 (PrrnLT7g10+DB/Ec; SEQ ID NO: 14) plastid(PrrnLT7g10+DB/pt; SEQ ID NO: 15) and synthetic DB (PrrnLT7g10−DB; SEQID NO: 16). For details see description of FIG. 3A.

FIG. 4A. Plastid transformation vector pPRV111A with chimeric neo genes.Plasmid serial numbers, for example pHK34, designate pPRV111A plastidtransformation vectors derivatives; adjacent plasmid numbers inparenthesis (e.g. pHK14) designate the source of the chimeric neo genein pUC118 or pBSIIKS+vectors. Arrows mark orientation of the selectablemarker gene (aadA) and of the chimeric neo gene. Plastid targetingsequences are underlined in bold. Components of the chimeric neo genesare: Prrn, rRNA operon promoter fragment; L, leader sequence; DB,downstream box; NheI site which serves as a synthetic DB is marked by aheavy line; neo, neomycin phosphotransferase coding region; TrbcL, rbcL3′-untranslated region. 16SrDNA, trnV, rps12/7 are plastid genes(Shinozaki et al., 1986). The restriction sites marked for: EcoRI, SphI,StuI, SacI, NheI, NcoI, XbaI, HindIII, BamHI and BglII. Restrictionsites in brackets were eliminated during construction. The neotranslation initiation in plasmid pHK36 is included in NcoI site (notmarked). The presence and relative order of NheI (**) and NcoI (*)restriction sites in the plasmid pPRV111A−DB derivatives (pHK35, pHK37,pHK40, pHK42, pHK43) are marked by asterisks. The promoter sequences areshown in FIGS. 3B, C and D.

FIG. 4B. Plastid transformation vector pPRV111B with chimeric neo genes.See description of FIG. 4A. The promoter sequences are shown in FIG. 3A.

FIG. 5. Construction of Prrn promoter-plastid leader fragments byoverlap extension PCR.

FIG. 6. Construction by the PCR of PrrnLT7g10+DB/Ec promoter (SacI-NheIfragment) in plasmid pHK18.

FIG. 7. Construction by PCR of the PrrnLT7g10+DB/pt promoter (SacI-NheIfragment) in plasmid pHK19.

FIG. 8. Restriction map of plasmids pHK2 and pHK3 with thePrrn(L)rbcL(S)::neo::TrbcL gene. Restriction enzyme cleavage sites aremarked for: BamHI, EcoRI, HindIII, NcoI, NheI, SacI, XbaI.

FIG. 9. DNA sequence of the Prrn(L)rbcL(S)::neo::TrbcL gene in plasmidPhk3 (SEQ ID NO: 17). Plasmid pHK2 carries an identical neo gene, exceptthat there is an EcoRI site upstream of the SacI site.

FIG. 10. NPTII accumulation in tobacco leaves detected by protein gelblot analysis. Amount of total soluble leaf protein (μg) loaded onSDS-PAGE gel is indicated above the lanes. Lanes are designated withplasmid used for plant transformation; μg protein loaded per lane isgiven below. NPTII standard and Nt-pTNH32 extracts were run as positivecontrols; extracts from wild-type non-transformed plants (wt) were usedas negative controls.

FIG. 11. The levels of neo mRNA in the transplastomic leaves. The blotswere probed for neo (top) and cytoplasmic 25S rRNA as loading control(bottom). Positions of the monocistronic neo mRNA in vector pPRV111A(FIG. 4A), the monocistronic neo and dicistronic neo-aadA transcripts invector pPRV111B (FIG. 4B) and the monocistronic neo and dicistronicrbcL-neo transcripts in pTNH32 transformed plants (Carrer et al., 1993)are marked. Lanes are designated with the transgenic plant serialnumber. 4 μg total cellular RNA was loaded per lane.

FIG. 12. Fraction of a codon encoding a particular amino acid andtriplet frequency per 1000 codons in the mutagenized atpB and rbcL DBregion (atpB wt: nucleotide sequence is nucleotides 1 through 42 of SEQID NO: 111, amino acid sequence is SEQ ID NO: 132; atpB m: nucleotidesequence is nucleotides 1 through 42 of SEQ ID NO: 112, amino acidsequence is SEQ ID NO: 132; rbcL wt: nucleotide sequence is nucleotides1 through 42 of SEQ ID NO: 114, amino acid sequence is SEQ ID NO: 122;rbcL m: nucleotide sequence is nucleotides 1 through 42 of SEQ ID NO:115, amino acid sequence is SEQ ID NO: 122; T7g10+DB/Ec: nucleotidesequence is SEQ ID NO: 123, amino acid sequence is SEQ ID NO: 124;T7g10+DB/pt: nucleotide sequence is SEQ ID NO: 125, amino acid sequenceis SEQ ID NO: 126; T7g10−DB: nucleotide sequence is SEQ ID NO: 127,amino acid sequence is SEQ ID NO: 128). Altered nucleotides are in lowercase.

FIG. 13A. NPTII accumulation in tobacco roots detected by protein gelblot analysis. Lanes are designated with the plasmid used for planttransformation; μg protein loaded per lane is given below. NPTIIstandard was run as positive control; extracts from wild-typenon-transformed plants (wt) were used as negative controls.

FIG. 13B. Steady-state levels of neo mRNA in tobacco roots. The neoprobe detects a monocistronic mRNA in plants transformed with vectorpPRV111A (FIG. 4A), and a monocistronic neo and a dicistronic neo-aadAtranscript in plants transformed with vector pPRV111B (FIG. 4B). Lanesare designated with the transgenic plant serial number. 4 μg totalcellular RNA was loaded per lane.

FIG. 14. Protein gel blot analysis to detect NPTII accumulation intobacco seeds. Lanes are designated with plasmid used for planttransformation; μg protein loaded per lane is given below. NPTIIstandard was run as positive control; extracts from wild-typenon-transformed plants (wt) were used as negative controls.

FIG. 15A. Diagram showing integration of the chimeric neo and aadA genesinto the plastid genome by two homologous recombination events via theplastid targeting sequences (underlined). On top is shown a diagram ofplasmids pHK30 and pHK32 are plastid transformation vector pPRV111Bderivatives (Zoubenko et al., 1994). Horizontal arrows mark geneorientation. For description of chimeric neo genes, see FIG. 4B.16SrDNA, trnV, rps12/7 are plastid genes (Shinozaki et al., 1986). Therestriction sites marked for: EcoRI (E), SacI (S), NheI (N), XbaI (X),HindIII (H), BamHI (Ba) and BglII Restriction sites in brackets wereeliminated during construction. In the middle the wild-type plastid DNAregion (Wt-ptDNA) targeted for insertion is shown. Lines connectingplasmids and ptDNA mark sites of homologous recombination at the end ofthe vector plastid-targeting regions. The transformed plastid genomesegment (T-ptDNA) map is shown on the bottom.

FIG. 15B. DNA gel blot analysis confirms of integration of the neo andaadA genes into the plastid genome. The blot on top was probed with theplastid targeting sequence (Probe 1 in FIG. 15A). It lights up 4.2-kband 1.4-kb fragments in transplastomic lines, and a 3.1-kb fragment inwild-type (see FIG. 15A). Note that the 1.4-kb signal is week in mostclones. The blot on the bottom was probed for neo sequences, which arepresent only in the transplastomic lines.

FIG. 16A. Diagram showing integration of the bar gene into the tobaccoplastid genome. Map of the plastid targeting region in plasmid pJEK6 isshown on top. The targeted region of the wild-type plastid genome(wt-ptDNA) is shown in the middle. Integrated transgenes in thetransplastome (T-ptDNA) are shown at the bottom. Map positions are shownfor: the bar gene; aadA, the selectable spectinomycin resistance gene;16SrDNA and rps12/7, plastid genes (Shinozaki et al.; 1986). Arrowsindicate direction of transcription. Map position of the probe (2.5 kb)is marked by a heavy line; the wild-type (2.9-kb) and transgenic(3.3-bk, 1.9-kb) fragments generated by SmaI and BglII digestion aremarked by thin lines.

FIG. 16B. DNA gel blot confirms integration of bar into tobacco plastidgenome. Data are shown for transplastomic lines Nt-pJEK6-2A through E,Nt-pJEK6-5A through E and Nt-pJEK6-13A and B, and the wild-type parentalline. SmaI-BglII digested total cellular DNA was probed with the 2.5-kbApaI-BamHI plastid targeting sequence marked with heavy line in FIG.16A.

FIG. 17. PAT assay confirms bar expression in tobacco plastids. PATactivity was determined by conversion of PPT into acetyl-PPT usingradiolabeled ¹⁴C-Acetyl-CoA. Data are shown for transplastomic linesNt-pJEK6-2D, Nt-pJEK6-5A and Nt-pJEK6-13B, nuclear transformantNt-pDM307-10 and wild-type (wt).

FIG. 18A. Transplastomic tobacco plants are herbicide resistant.Wild-type and pJEK6-transformed plants 13 days after Liberty spraying (5ml, 2% solution).

FIG. 18B. Maternal inheritance of PPT resistance in the seed progeny.Seeds from reciprocal crosses with Nt-pJEK6-5A plants germinated on 0,10 and 50 mg/L PPT. wt×pJEK6-5A, transplastomic used as pollen parent;pJEK6-5A×wt, transplastomic line female parent. Resistant seedlings aregreen on PPT medium, sensitive seedlings are bleached.

FIG. 19. The engineered bacterial bar coding region DNA sequence inplasmid pJEK3 and pJEK6 (SEQ ID NO: 18) and encoded amino acid sequence(SEQ ID NO: 129). Nucleotides encoding the rbcL five N-terminal aminoacids are in lower case. Nucleotides added at the 3′ end duringconstruction are also in lower case. NcoI, BglII and XbaI cloning sitesare marked.

FIG. 20A. The synthetic bar gene DNA sequence (SEQ ID NO: 19) and theencoded amino acid sequence (SEQ ID NO: 130). The arginines encoded byAGA/AGG codons are in bold. Original nucleotides are in capital letters,altered bases are in lower case. Restriction sites used for cloning aremarked.

FIG. 20B. The synthetic s2-bar gene DNA sequence (SEQ ID NO: 20) and theencoded amino acid sequence (SEQ ID NO: 130). The arginines encoded byAGA/AGG codons are in bold. Original nucleotides are in capital letters,altered bases are in lower case. Restriction sites used for cloning aremarked.

FIG. 21. Synthetic and bacterial bar genes. The bar coding region isexpressed in the Prrn/TrbcL cassettes. Note that the Prrn promotersdiffer with respect to the translational control region.

FIG. 22A. PAT is expressed in E. coli from bar, but not from s-barcoding region. PAT activity was determined by conversion of PPT intoacetyl-PPT using radiolabeled ¹⁴C-Acetyl-CoA. Data are shown for E. colitransformed with plasmids pJEK6 and pKO12 carrying the bar gene, andpKO8, carrying s-bar.

FIG. 22B. PAT assay confirms expression of bar and s-bar in tobaccoplastids. PAT activity was determined by conversion of PPT intoacetyl-PPT using radiolabeled ¹⁴C-Acetyl-CoA. Data are shown fortransplastomic lines Nt-pJEK6-13B and Nt-pKO3-24a,B carrying bar ands-bar, respectively.

FIG. 23A. Plastid transformation vector with FLARE16-S as selectablemarker targeting the plastid inverted repeat region. DNA (SEQ ID NO:131) and protein (SEQ ID NO: 104) sequence at the aadA-gfp junction.Nucleotides derived from aadA and gfp are in capital, adapters sequencesand the point mutation used to create the BstXI restriction site (bold)are in lower case.

FIG. 23B. Physical map of plastid transformation vector with FLARE16-Sas selectable marker targeting the plastid inverted repeat region. Shownare: the promoter (P) and 3′UTR (T) of the aadA16pt-gfp coding regionand its component parts (aadA and gfp coding regions); rrn16 and rps12/7plastid genes; restriction endonuclease sites HindIII (removed), SpeI,XbaI, NcoI, BstXI, NheI, EcoRI. In plasmid pMSK56 aadA16pt-gfp isexpressed from the Prrn:LatpBDB promoter and encodes FLARE16-S1. Inplasmid pMSK57 aadA16pt-gfp is expressed from the Prrn:LrbcLDB promoterand encodes FLARE16-S2.

FIG. 24. Localization of FLARE16-S to tobacco plastids by laser scanningconfocal microscopy in heteroplastomic tissue. Images were processed todetect FLARE16-S (green) and chlorophyll fluorescence (red) and both ina merged view. Sections are shown from plants expressing FLARE16-S1(a,b) and FLARE16-S2 (3c-f). Note wild-type and transformed plastids inleaves (3a,c,d), chromoplasts of petals (3b), trichomes (3e) andnon-green root plastids (f). White arrows mark transplastomicorganelles. Bars represent 25 μm.

FIG. 25. Immunoblot analysis of FLARE16-S accumulation in chloroplasts.The amount of loaded protein (μg) is indicated above the lanes.Quantification of FLARE16-S1 (Nt-pMSK56 plants) and FLARE16-S2(Nt-pMSK57 plants) is based on comparison with a purified GFP dilutionseries. Extract from a wild-type plant (Nt) was used as negativecontrol.

FIG. 26A. Amplification of border fragments confirms integration ofFLARE-S genes into the plastid genome. Maps of the plastid targetingregions of the rice (pMSK49) and tobacco (pMSK57) vectors, the segmentof the rice and tobacco plastid genomes targeted by the vectors (Os-wtand Nt-wt), and the same regions after integration of FLARE-S genes. Theends of plastid targeting regions are connected with cognate sequencesin the wild-type plastid genome. Plastid genes 16SrDNA, trnV and rps12/7are marked only in the wild-type plastid genomes. The position of PCRprimers (01-06) and the PCR fragments generated by them are also shown.

FIG. 26B. Amplification of border fragments confirms integration ofFLARE-S genes into the plastid genome. Gels with PCR-amplified left andright border fragments, and with aadA fragment. Results are shown forrice (Os-pMSK49-1 and Os-pMSK49-2) and tobacco (Nt-pMSK57)transplastomic lines and wild-type (Os-wt) rice. The molecular weightmarkers is EcoRI- and HindIII-digested λ DNA.

FIG. 27. Localization of FLARE11-S3 to rice chloroplasts in theOs-pMSK49-5 line by laser scanning confocal microscopy. Images wereprocessed to detect FLARE11-S (green) and chlorophyll fluorescence (red)and both in a merged view. Arrows point to mixed populations of plastidsin cells. Bar represents 25 μm.

FIG. 28. The sequence of FLARE16-S is shown (SEQ ID NO: 21).

FIG. 29. The sequence of FLARE16-S1 is shown (SEQ ID NO: 22).

FIG. 30. The sequence of FLARE16-S2 is shown (SEQ ID NO: 23).

FIG. 31. The sequence of FLARE11-S is shown (SEQ ID NO: 24).

FIG. 32. The sequence of FLARE11-S3 is shown (SEQ ID NO: 25).

FIGS. 33A and 33B. The sequence of pMSK35 is shown (SEQ ID NO: 26).

FIGS. 34A and 34B. The sequence of pMSK49 is shown (SEQ ID NO: 27).

FIG. 35. A table describing the FLARE constructs of the invention.

DETAILED DESCRIPTION OF THE INVENTION

DNA cassettes for high level protein expression in plastids are providedherein. Higher plant plastid mRNAs contain sequences within 50 ntdownstream of AUG that are complementary to the 16S rRNA 3-region. Thesecomplementary sequences are approximately at the same position as DBsequences in E. coli mRNAs. See FIGS. 1A and 2A. Interestingly, thetentative plastid DB sequence significantly deviates from the E. coli DBconsensus, since the tobacco plastid and E. coli 16S rRNA sequence inthe anti-downstream-box (ADB) region is significantly different (FIG.1B). The feasibility of improving protein expression by incorporating DBsequences in plastids was assessed by constructing a series of chimeric5′ regulatory regions consisting of the plastid rRNA operon σ⁷⁰-typepromoter (Prrn-114; Svab and Maliga, 1993; Vera and Sugiura, 1995) andthe leader sequence of plastid mRNAs with the native DB, mutagenized DBand synthetic DB sequences. The plastid mRNA leaders differ with respectto the presence and position of the SD sequence. Translation efficiencyfrom the chimeric promoters was determined by expressing the bacterialneo gene in plastids. The neo (or kan) gene encodes neomycinphosphotransferase (NPTII) and confers resistance to kanamycin inbacteria and plastids (Carrer et al., 1993). We have found that NPTIIfrom the chimeric neo transcripts accumulates in the range of 0.2% to23% of the total soluble leaf protein, indicating the importance oftranslational control signals in the mRNA 5′ region for high-levelprotein expression.

There is great interest in producing recombinant proteins in plantsplastids which, thus far have been expressed from nuclear genes only(Arntzen, 1997; Conrad and Fiedler, 1998; Kusnadi et al., 1997). Proteinlevels produced from the PrrnLrbcL+DBwt and PrrnLT7g10 expressioncassettes described here significantly exceed protein levels reportedfor nuclear genes. Accumulation of NPTII from nuclear genes is typically<<0.1% (Allen et al., 1996), the highest value being 0.4% of the totalsoluble protein (Houdt et al., 1997). We reported earlier accumulationof 1% NPTII from a plastid neo transgene (Carrer et al., 1993). Otherexamples for protein accumulation from plastid transgenes are 2.5%β-glucuronidase (GUS) (Staub and Maliga, 1993)) and 3-5% of the Bacillusthuringiensis (Bt) crystal toxins (McBride et al., 1995). As compared tothis earlier report, we have achieved a significant increase in NPTIIlevels, up to 23% of total soluble protein.

FLARE-S, a protein obtained by fusing an antibiotic-inactivating enzymewith the Aequorea victoria green fluorescence protein accumulated to 8%and 18% of total soluble protein from the PrrnLatpB+DBwt andPrrnLrbcL+DBwt cassettes provided herein. See Example 8. High-levelprotein accumulation from the cassettes of the present invention can beclearly attributed to engineering the translational control region (TCR)of the chimeric genes. These novel genetic elements may be used indifferent applications to drive expression of proteins with agronomic,industrial or pharmaceutical importance.

There is a strong demand for methods that control the flow of transgenesin field crops. Incorporation of the transgenes in the plastid genomerather than the nuclear genome results in natural transgene containment,since plastids are not transmitted via pollen in most crops (Maliga,1993). Plastid transformation in crops has not been widely employed dueto the lack of technology. Enhanced expression of selective markersshould yield higher transformation efficiencies. The chimeric promotersof the present invention facilitate extension of plastid transformationto agronomically and industrially important crops. Indeed, high-levelexpression from the PrrnLatpB+DBwt cassette described here resulted in˜25-fold increase in the frequency of kanamycin-resistant transplastomictobacco lines. More importantly, high levels of marker gene expressionfollowing plastid transformation have been obtained in rice, the firstcereal species in which plastid transformation has been successful. Theresults are set forth in Example 8.

The following examples are provided to illustrate various embodiments ofthe present invention. They are not intended to limit the invention inany way.

The protocols set forth below are provided to facilitate the practice ofthe present invention.

Preparation of Chimeric 5′ Cassettes for Elevated Expression ofHeterologous Proteins in Plastids of Higher Plants

Identification of a Potential Downstream Box in Plastid mRNAs

The presence or absence of downstream box elements in mRNA molecules wasdetermined for the following genes: psbB (Tanaka et al., 1987) and psbA(Sugita and Sugiura, 1984), photosystem II genes; rbcL, encoding thelarge subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase(Shinozaki and Sugiura, 1982); atpB, encoding the ATPase β subunit(Orozco et al., 1990); and clpP, encoding the proteolytic subunit of theClp ATP-dependent plastid protease (Hajdukiewicz et al., 1997).Interestingly, most or all of the PclpP-53 promoter is downstream of thetranscription initiation site, therefore the PrrnLclpP constructs areassumed to contain two promoters: Prrn-114 and PclpP-53. Transcriptioninitiation sites for these genes were described in references citedabove; for nucleotide position of the genes in the plastid genome seeShinozaki et al., 1986.

Initially, it was assumed that the plastid ADB is similar in size andposition as the E. coli ADB in the 16S rRNA. The E. coli ADB islocalized on a conserved stem structure between nucleotides 1469 to 1483(15 nt) that corresponds to nucleotides 1416 and 1430 of the plastid 16SrRNA (Dams et al., 1988; Sprengart et al., 1996). Although in bothcases, the ADB is contained in the 16S rRNA penultimate stem, the actualADB sequence is different in plastids and in E. coli (FIG. 1B). TheN-terminal coding regions of plastid genes atpB, clpP, rbcL, petA, psaA,psbA, psbB, psbD and psbE were searched for potential DB sequences. Thehomology search was carried out with a 26 nucleotide sequence centeredon the tentative DB region (FIG. 1B). The search revealed shortstretches of imperfect homology with alternative solutions. Since theposition of DB in the mRNA is quite flexible (Etchegaray and Inouye,1999), we show four potential DB-ADB interactions for atpB and rbcL inFIG. 2A. Two plastid mRNAs were selected to test the role of DB in thetranslation of plastid mRNAs: 1) atpB mRNA lacks a SD sequence; and 2)rbcL mRNA contains a SD sequence at the prokaryotic consensus. Inaddition, the phage T7 gene 10 (T7g10) leader was included in the study.This leader has a well-characterized E. coli DB sequence (FIG. 2B;Sprengart et al., 1996). Additional plastid mRNAs with potential DBsequences shown in FIG. 2A are clpP, psbB and psbA.

Experimental Strategy to Test the Efficiency of Leader Sequences forTranslation

To compare the efficiency of translation from the 5′-UTR of the selectedgenes, the 5′-UTR was cloned downstream of the strong plastid rRNAoperon σ⁷⁰-type promoter (Prrn-114) (Svab and Maliga, 1993; Allison etal., 1996), which initiates transcription from multiple adjacentnucleotides (−114, −113, −111; Sriraman et al., 1998). The promoterfragments were constructed as SacI-NheI or a SacI-NcoI fragments.Construction of the chimeric promoters using conventional molecularbiological techniques is set forth in detail in the next section.

Two constructs were prepared for each 5′-UTR selected: one with (+DB)and one without (−DB) a native downstream box. It will be obvious fromthe forthcoming discussion, that the −DB constructs have a synthetic DBprovided by the NheI restriction site. The promoters were clonedupstream of the coding region of a kanamycin resistance (neo) gene,which is available on an NheI-XbaI or NcoI-XbaI fragment. For thestabilization of the mRNA, the rbcL gene 3′-untranslated region wascloned downstream of neo as an XbaI-HindIII fragment. The chimeric neogenes can therefore be excised from the pUC118 or pBSIIKS+plasmids asSacI-HindIII fragments. These source plasmids are listed in Table 1.TABLE 1 Salient features of chimeric promoters^(a.) Source of 5′-UTRPromoter pUC118(U) or (nucleotides from AUG) SD DB fragment pBSIIKS⁺(B)pPRV111A, B atpB (−90/+42) − wt SacI/NheI pHK10(U) pHK30(B) atpB(−90/+6) − s SacI/NheI pHK11(U) pHK31(B) atpB (−90/42) − m SacI/NheIpHK50(B) pHK60(B) clpP (−53/+48) − wt SacI/NheI pHK12(U) pHK32(B) clpP(−53/+6) − s SacI/NheI pHK13(U) pHK33(B) rbcL (−58/+42) + wt SacI/NheIpHK14(B) pHK34(A) rbcL (−58/+6) + s SacI/NheI pHK15(U) pHK35(A) rbcL(−58/+42) + m SacI/NheI pHK54(B) pHK64(A) psbB (−54/+45) + wtSacI/NheI^(d) pHK16(U) pHK36(A) psbB (−54/+3) + s SacI/NcoI^(d) PHK17(U)pHK37(A) ^(b)T7g10+DB/Ec (−63/+24) + Ec SacI/NheI pHK18(B) pHK38(A)^(b)T7g10+DB/pt (−63/+24) + pt SacI/NheI pHK19(B) pHK39(A) T7g10−DB(−63/+9) + s SacI/NheI pHK20(B) pHK40(A) psbA (−85/+21) − wt SacI/NheIpHK21(U) pHK41(A) psbA (−85/+3) − s SacI/NcoI^(e) pHK22(U) pHK42(A)^(c)psbA(+GC) (−85/+3) − s SacI/NcoI^(e) pHK23(U) pHK43(A)^(a)SD+, SD at prokaryotic consensus position; SD−, no SD at prokaryoticconsensus position; DB wt, wild-type; m, mutants; s, NheI site assynthetic DB.^(b)Ec or pt refers to construct with E. coli or plastid DB sequence.^(c)psbA(+GC) indicates addition of GC to the wild-type A at the mRNA5′-end.^(d)In source gene psbB translation initiation codon is within NcoIsite; therefor +DB construct pHK16 has this NcoI site upstream of theNheI site; see FIG. 9.^(e)Translation initiation codon is included in NcoI site; NheI site isdirectly downstream in kan coding region; see FIG. 8.

The Prrn promoter fragment is available in plasmid pPRV100A (Zoubenko etal., 1994). The promoters were designed to include sequences between−197 nt and −114 nt upstream of the mature 16S rRNA 5′ end. Nucleotide−197 is the 5′-end of the Prrn promoter constructs utilized for theseand other studies (Svab and Maliga. 1993; −1 is the first nucleotideupstream of the mature 16S rRNA). The G at the −114 position is one ofthree transcription initiation sites; the other two are the adjacent C(−113) and A (−111) nucleotides (Allison et al., 1996, Sriraman et al.,1998). The nucleotide at which Prrn transcription would initiate ismarked by a filled circle in FIG. 3A-D. In most constructs, this is a G(−114) as in the native promoter. In two constructs the G was replacedby an A, as in the psbA promoter which is the source of the leadersequence (pHK21, pHK22; see below).

Design of the 5′ Leader from atpB

For the atpB gene, multiple mRNA 5′-ends were mapped in tobacco leavesincluding at least four primary transcripts indicating transcriptionfrom four promoters and a processed 5′-end 90 nucleotides upstream ofthe translation initiation codon (Orozco et al., 1990). The terminalnucleotide of the processed atpB 5′-end is a G. Therefore, the chimericPrrnLatpB promoters were designed to initiate transcription at a G,anticipating that the leader sequence of the chimeric transcript will bea perfect reproduction of the processed atpB mRNA 5′-end. Out of theatpB coding region, 42 and 6 nucleotides are included in the +DBwt and−DB constructs, respectively. The 42 nucleotides include four potentialDB sequences shown in FIG. 2A. Two point mutations in the leadersequence were designed to eliminate NheI (T to A) and EcoRI (G to A)restriction sites without affecting the predicted mRNA 5′ secondarystructure. In the −DB constructs, two codons (6 nucleotides) wereretained from the native coding region upstream of the NheI restrictionsite (GCTAGC sequence) in which the stop codon is out-of-frame (FIG.3A). Eleven silent point mutations were introduced in the DB region ofthe PrrnLatpB+DBm construct to either minimize the number of base pairs,or to change the nature of base pairing (for example G-C to G-U) (FIG.2A; FIG. 3A).

Design of the 5′ Leader from clpP

Two major mRNA 5′-ends of the clpP gene were mapped in tobacco leaves(Hajdukiewicz et al., 1997). The terminal nucleotide of the proximalprimary transcript is a G. Therefore, the chimeric PrrnLclpP promoterswere designed to initiate transcription at a G, anticipating that theleader sequence of the chimeric transcript will be a perfectreproduction of the leader transcribed from the Pclp-53 NEP promoter.Out of the clpP coding region, 48 and 6 nucleotides are retained in the+DBwt and −DB constructs, respectively. The 48 nucleotides include fourpotential DB sequences as shown in FIG. 2A. In the −DB constructs, twocodons (6 nucleotides) were retained from the native coding regionupstream of the NheI restriction site (GCTAGC sequence) in which thestop codon is out-of-frame.

Design of the 5′ Leader from rbcL

One primary and one processed mRNA 5′-end were mapped in tobacco leavesfor the rbcL gene (Shinozaki and Sugiura, 1982). The terminal nucleotideof the processed 5′ end is a T. The chimeric PrrnLrbcL promoters weredesigned to initiate transcription at a G, one nucleotide downstream ofthe terminal T. Forty-two and 6 nucleotides out of the rbcL codingregion are included in the +DB and −DB constructs, respectively. The 42nucleotides include four potential DB sequences as shown in FIG. 2A. Theone point mutation (G to A) in the leader sequence was designed toeliminate an EcoRI restriction site without affecting the predicted mRNA5′ secondary structure. In the −DB constructs, two codons (6nucleotides) were retained from the native coding region upstream of theNheI restriction site (GCTAGC sequence) in which the stop codon isout-of-frame. Twelve silent point mutations were introduced into the DBregion of the PrrnLrbcL+DBm construct to either minimize the number ofbase pairs, or to change the nature of base pairing (for example G-C toG-U) (FIG. 2A, FIG. 3B).

Design of the 5′ Leader from psbB

One primary and one processed mRNA 5′-end for the psbB gene weretentatively identified in tobacco leaves (Tanaka et al., 1987). Theleader sequence was designed to initiate transcription from the G (−114)of the Prrn promoter, and include the intact secondary (stem) structureassumed to be involved in stabilizing the mRNA. Forty-five and 3nucleotides out of the psbB coding region are included in the +DB and−DB constructs, respectively. The 45 nucleotides include four potentialDB sequences shown in FIG. 2A. Since the ATG is naturally included in anNcoI site that is used to fuse the neo coding region with the psbBleader, no amino acid from the psbB coding region is added in the −DBconstruct.

Design of the 5′ Leader from psbA

One mRNA 5′-end was mapped for the psbA gene in tobacco leaves (Sugitaand Sugiura, 1984). The terminal nucleotide of the primary transcript isan A. Therefore, the chimeric PrrnLpsbA promoters were designed toinitiate transcription at an A, anticipating that the leader sequence ofthe chimeric transcript will be a perfect reproduction of the leadertranscribed from the psbA promoter. Twenty-one and 3 nucleotides out ofthe psbA coding region are included in the +DB and −DB constructs,respectively. The 21 nucleotides include the potential DB sequence asshown in FIG. 2A. Since the neo coding region was linked to the chimericpromoter via an NcoI site which includes the translation initiationcodon (ATG), no amino acid from the psbA coding region is added in the−DB constructs. This is true of a second −DB promoter, in plasmid PHK23,in which transcription is designed to initiate from the Prrn G (−114)and C (−113) (FIG. 3C).

Design of the T7 Phage Gene 10 Leader

The T7 phage gene 10 leader (63 nucleotides) was shown to promoteefficient translation initiation in E. coli (Olins et al., 1988). Thisleader is used in the E. coli pET expression vectors (Studier et al.,1990; Novagen Inc.). The terminal nucleotide at the 5′-end is a G.Therefore, the chimeric PrrnT7g10L promoters were designed to initiatetranscription at a G, anticipating that the leader sequence of thechimeric transcript will be a reproduction of the T7 phage gene 10 mRNA,with the exception of a T to A mutation which was introduced toeliminate an XbaI site. Twenty-four and 9 nucleotides from the T7 phagegene 10 coding region are included in the +DB/Ec (with E. coli DBsequence) and −DB constructs, respectively. To compare the efficiency ofE. coli and plastid DB sequences in plastids, a second +DB promoter wasconstructed with the tobacco DB sequence (PrrnT7g10L+DB/pt). The nativeT7g10 leader has an NheI site directly downstream of the translationinitiation codon. This NheI site was removed by a T to A point mutationin the +DB constructs (FIG. 3D).

For introduction into the plastid genome, the chimeric neo genes werecloned into plastid transformation vector pPRV111A or pPRV111B. See U.S.Pat. No. 5,877,402, the disclosure of which is incorporated herein byreference. The pPRV111 vectors target insertions into the invertedrepeat region of the tobacco plastid genome, and carry a selectablespectinomcyin (aadA) resistance gene. The sequences of the vectors havebeen deposited in GenBank (U12812, U12813). The chimeric neo gene invector pPRV111B is in tandem with the aadA gene, whereas in vectorpPRV111A the chimeric neo is oriented divergently. The general outlineof the plastid transformation vector with the chimeric neo genes isshown in FIGS. 4A and 4B.

Construction of Chimeric Prnn Promoters with Plastid mRNA Leaders

The chimeric Prrn promoter/leader fragments were constructed as aSacI-NheI or SacI-NcoI fragments (Table 1, below) by overlap extensionPCR (SOE-PCR), essentially as described in Lefebvre et al., (1995).Construction of the Prrn-plastid leader segments is schematically shownin FIG. 5. The objective of the PCR-1 step is to 1) amplify the Prrnpromoter fragment while 2) adding a SacI site upstream and a seam-lessoverlap with the specific downstream leader sequence. The reactioncontains: 1) a primer (oligonucleotide) to add a SacI site at the 5′-endof the fragment; 2) a suitable template containing the Prrn promotersequence in plasmid pPRV100A (Zoubenko et al., 1994); and 3) a primer toadd on the overlap with the leader sequence at the 3′ of the amplifiedproduct. The objective of the PCR-2 step is to create the chimericpromoter with DB sequence using: 1) the product of PCR-1 step as aprimer; 2) a suitable DNA template containing the specific leadersequence; and 3) primer (oligonucleotide) to include NheI restrictionsite at the 3′-end of the amplification product. The product of thePCR-2 is the SacI-NheI chimeric Prrn promoter fragment with DB sequence.The objective of the PCR-3 step is to remove the DB sequence whileintroducing a suitable NheI or NcoI restriction site. The product ofPCR-3 is the SacI-NheI or SacI-NcoI chimeric Prrn promoter fragment inwhich the DB sequence is replaced with the NheI site. The objective ofthe PCR-4 step is to replace the wild-type DB with a mutant DB. Theproduct of PCR-4 is a SacI-NheI Prrn promoter fragment.

The primers (oligonucleotides) used for the construction of chimericpromoters are listed in Table 2. The chimeric promoters were obtained byoverlap extension PCR using oligonucleotides and DNA templatesschematically shown in FIG. 5. TABLE 2 Oligonucleotides used for theconstruction of chimeric promoters.  #1:5′-CCCGAGCTCGCTCCCCCGCCGTCGTTC-3′ (SEQ ID NO:31)  #2:5′-CGAATTTAAAATAAATGTCCGCTTGCAC (SEQ ID NO:32)GTCGATCGGTTAATTCTCCCAGAAATATAGC CATCC-3′  #3:5′-CCCGCTAGCCGTGGAAACCCCAGAACC-3′ (SEQ ID NO:33)  #4:5′-CCCGCTAGCTCTCATAATAATAAAATAAAT (SEQ ID NO:34) AAATATGTC-3′  #5:5′-TCACTTTGAGGTGGAAACGTAACTCCCAGA (SEQ ID NO:35) AATATAGCCATCC-3′  #6:5′-CCCGCTAGCTTCCTCTCCAGGACTTCG-3′ (SEQ ID NO:36)  #7:5′-CCCGCTAGCAGGCATTAAATGAAAGAAAGA (SEQ ID NO:37) AC-3′  #8:5′-TAAGAATTTTCACAACAACAAGGTCTACTC (SEQ ID NO:38)GACTCCCAGAAATATAGCCATCC-3′  #9: 5′-CCCGCTAGCTTTGAATCCAACACTTGCTTT (SEQID NO:39) AG-3′ #10: 5′-CCCGCTAGCTGACATAAATCCCTCCCTAC- (SEQ ID NO:40) 3′#11: 5′-CAAAGATAAATAGACACTACGTAACTTTAT (SEQ ID NO:41)TGCATTGCTCCCAGAAATATAGCCATCC-3′ #12: 5′-CCCGCTAGCATCATTCAATACAACGGTATG(SEQ ID NO:42) AACACG-3′ #13: 5′-TTCTAGTGGGAAACCGTTGTGGTCTCCCTC (SEQ IDNO:43) CCAGAAATATAGCCATCC-3′ #14: 5′-CCCGCTAGCCATATGTATATCTCCTTCTTA (SEQID NO:44) AAG-3′ #15: 5′-CCCGCTAGCCTGTCCACCAGTCATGCTTGC (SEQ ID NO:45)CATA-3′ #16: 5′-CCCGCTAGCCAAGGCAGGGCTAGTGATTGC (SEQ ID NO:46)CATATGTATATCTCCTTC-3′ #17: 5′-TTTGTTTAACTTTAAGAAGGAGATATACAT (SEQ IDNO:47) ATGGCAAOCATGACTGGTGG-3′ #18: 5′-CTCCTTCTTAAAGTTAAACAAAATTATTTC(SEQ ID NO:48) TAGTGGGAAACCGTTGT-3′ #19:5′-CAAAATAGAAAATGGAAGGCTTTTTGCTCC (SEQ ID NO:49) CAGAAATATAGCCATCCC-3′#20: 5′-CAAAATAGAAAATGGAAGGCTTTTTTCCCA (SEQ ID NO:50)CAAATATAGCCATCCC-3′ #21: 5′-GGGCCATGGTAAAATCTTGCTTTATTTAAT (SEQ IDNO:51) C-3′ #22: 5′-GGGGCTAGCTCTCTCTAAAATTGCAGT-3′ (SEQ ID NO:52) #23:5′-GAATAGCCTCTCCACCCA-3′ (SEQ ID NO:53) #24:5′-CCCGCTACCCGTGGACACCCCACTTCCACT (SEQ ID NO:54)TGTTGTCGGGTTTATTCTCAT-3′ #25: 5′-CCCGCTAGCTTTGAATCCTACTGAGGCTTT (SEQ IDNO:55) TGTTTCTGTTTGAGGACTCAT-3′Construction of Chimeric Prnn Promoter/atpB Leader SegmentsPrrnLatpB+DBwt in plasmid pHK10 (Product of PCR-2) PrrnLatpB−DB inplasmid pHK11 (Product of PCR-3) PrrnLatpB+DBm in plasmid pHK50 (Productof PCR-4)PCR-1: Oligonucleotides #1, #2 as primers; plasmid pPRV100A (Zoubenko etal., 1994) DNA as template.PCR-2: Product of PCR-1 step, Oligonucleotide #3 as primers; plasmidpIK79 (see below) DNA as template.PCR-3: Oligonucleotide #1, #4 as primers; Product of PCR-2 step astemplate.PCR-4: Oligonucleotide #1, #24 as primers; Product of PCR-2 step astemplate.Plasmid pIK79 is a Bluescript BS+phagemid derivative which carries aPvuII/XhoI tobacco plastid DNA fragment between nucleotides 55147-60484containing the rbcL-atpB intergenic region with divergent promoters forthese genes (Shinozaki et al., 1986).Construction of Chimeric Prnn Promoter/clpP Leader SegmentsPrrnLclpP+DBwt in plasmid pHK12 (Product of PCR-2) PrrnLclpP−DB inplasmid pHK13 (Product of PCR-3)PCR-1: Oligonucleotides #1, #5 as primers; plasmid pPRV100A (Zoubenko etal., 1994) DNA as template.PCR-2: Product of PCR-1 step, Oligo #6 as primers; tobacco Sal8 ptDNAfragment (Shinozaki et al., 1986) as template.PCR-3: Oligonucleotide #1, #7 as primers; Product of PCR-2 step astemplate.Construction of Chimeric Prnn Promoter/rbcL Leader SegmentsPrrnLrbcL+DBwt in plasmid pHK14 (Product of PCR-2) PrrnLrbcL−DB inplasmid pHK15 (Product of PCR-3) PrrnLrbcL+DBm in plasmid pHK54 (Productof PCR-4)PCR-1: Oligonucleotides #1, #8 as primers; plasmid pPRV100A (Zoubenko etal., 1994) DNA as template.PCR-2: Product of PCR-1 step, Oligonucleotide #9 as primers; plasmidpIK79 DNA (see description of pHK10 above) as template.PCR-3: Oligonucleotide #1, #10 as primers; Product of PCR-2 step astemplate.PCR-4: Oligonucleotide #1, #25 as primers; Product of PCR-2 step astemplate.Construction of Chimeric Prnn Promoter/psbB Leader SegmentsPrrnLpsbB+DBwt in plasmid pHK16 (Product of PCR-2) PrrnLpsbB−DB inplasmid pHK17 (Promoter from pHK16, digested with SacI/NcoI)PCR-1: Oligonucleotides #1, #11 as primers; plasmid pPRV100A (Zoubenkoet al., 1994) DNA as template.PCR-2: Product of PCR-1 step, Oligo #12 as primers; tobacco Sal8 ptDNAfragment (Shinozaki et al., 1986) as template.PCR-3 was not necessary, since the psbB translation initiation codon isnaturally included in an NcoI site. Therefore, the −DB derivative couldbe obtained by SacI/NcoI digestion of the PCR-2 step.Construction of Chimeric Prnn Promoter/psbA Leader SegmentsPrrnLpsbA+DBwt in plasmid pHK21 (Product of PCR-2) PrrnLpsbA −DB inplasmid pHK22 (Product of PCR-3)PCR-1: Oligonucleotides #1, #20 as primers; plasmid pPRV100A (Zoubenkoet al., 1994) DNA as template.PCR-2: Product of PCR-1 step, Oligo #22 as primers; tobacco Sal3 ptDNAfragment (Shinozaki et al., 1986) as template.PCR-3: Oligonucleotide #1, #21 as primers; Product of PCR-2 step astemplate.PrrnLpsbA(GC)−DB in plasmid pHK23 (Product of PCR-2)PCR-1: Oligonucleotides #1, #19 as primers; plasmid pPRV100A (Zoubenkoet al., 1994) DNA as template.PCR-2: Product of PCR-1 step, Oligo #21 as primers; tobacco Sal3 ptDNAfragment (Shinozaki et al., 1986) as template.

In all of the above, PCR amplification was carried out with AmpliTaq DNApolymerase (Perkin Elmer) or Pfu DNA polymerase (Stratagene) and“stepdown” PCR that utilizes gradually decreasing annealing temperatureswas performed (Hecker and Roux, 1996). The exact amplificationconditions for the chimeric Prrn::LatpB promoters are given below. Theamplification conditions for the remaining chimeric Prrn—plastid leaderpromoters were calculated according to Hecker and Roux (1996), anddiffer only in the annealing temperatures. Description of PCR conditionsfor the construction of the chimeric Prrn promoters with plastid mRNAleaders is given below; for interpretation of individual steps seescheme in FIG. 5.

PCR-1 Program: 50 picomoles of both primers per 100 μl 1.1 Denature   5min. at 94° C. 2.1 Denature   1 min. at 94° C. 3 cycles 2.2 Annealing0.5 min. at 72° C. 2.3 Extension 0.5 min. at 72° C. 3.1 Denature   1min. at 94° C. 3 cycles 3.2 Annealing 0.5 min. at 69° C. 3.3 Extension0.5 min. at 72° C. 4.1 Denature   1 min. at 94° C. 3 cycles 4.2Annealing 0.5 min. at 66° C. 4.3 Extension 0.5 min. at 72° C. 5.1Denature   1 min. at 94° C. 3 cycles 5.2 Annealing 0.5 min. at 63° C.5.3 Extension 0.5 min. at 72° C. 6.1 Denature   1 min. at 94° C. 3cycles 6.2 Annealing 0.5 min. at 60° C. 6.3 Extension 0.5 min. at 72° C.7.1 Denature   1 min. at 94° C. 20 cycles  7.2 Annealing 0.5 min. at 57°C. 7.3 Extension 0.5 min. at 72° C. 8.1 Extension  10 min. at 72° C. 8.2  1 min. at 30° C.

The PCR-2 program was essentially identical to the PCR1 program setforth above with the following modifications: 1) Primers in 100 μl werethe products of 1st PCR reaction, 50 picomoles of the oligonucleotideprimer were used; and 2) the annealing temperature in stepdown PCR wasfrom 67° C. to 52° C. Accordingly, the following annealing temperatureswere used: Step 2.2, 67° C.; Step 3.2, 64° C.; Step 4.2, 61° C.; Step5.2, 58° C.; Step 6.2, 55° C.; Step 7.2, 52° C.

The PCR-3 and PCR-4 programs were essentially identical to the PCR1program with the following modification: 1) The annealing temperature instepdown PCR was from 69° C. to 44° C. Accordingly, the followingannealing temperatures were used: Step 2.2, 69° C.; Step 3.2, 64° C.;Step 4.2, 59° C.; Step 5.2, 54° C.; Step 6.2, 49° C.; Step 7.2, 44° C.In cases where the yield of the final PCR reaction was too low forefficient cloning, final product was amplified using primers which wereused to generate the ends. The final PCR products were digested with theappropriate restriction enzymes (SacI and NheI or SacI and NcoI) andcloned in plasmids pHK2 or pHK3 (see below).

Construction of Chimeric Promoters with T7 Phage Gene 10 mRNA LeaderSegment

The chimeric Prrn promoter/T7gene10 leader (PrrnLT7g10) fragments wereconstructed as SacI-NheI fragments (Table 1, below).

PrrnLT7g10+DB/Ec Promoter in Plasmid pHK18

In the absence of a proper DNA template, the PrrnLT7g10+DB/Ec wasconstructed by employing a modified polymerase chain reaction (Uchida,1992) in two PCR steps, as schematically shown in FIG. 6. The PCR-1A andPCR1B steps generate two fragments in two separate reactions (A and B).The objective of the PCR-1A step is to amplify Prrn promoter fragmentwhile: 1) adding a SacI site upstream (Oligonucleotide #1 in Table 2);and 2) a seam-less overlap with the specific downstream leader sequence(Oligonucleotide #13 in Table 2) using plasmid pPRV100A (Zoubenko etal., 1994) as DNA template. The objective of the PCR-1B step is toamplify part of the T7g10 leader sequence using overlappingoligonucleotides #15 and #17 in Table 2. The NheI site is introduced inoligonucleotide #15. Both PCR-1A and PCR-1B reactions were carried outby stepdown PCR as described above for the construction of the chimericPrrn promoters.

PCR-2 reaction generating this chimeric promoter contained:

a) The products of the PCR-1A and PCR-1B reactions as DNA templates;

b) Oligonucleotide #18 (0.5 picomole; Table 2) to generate overlappingfragments with products of the PCR-1A and PCR-1B reactions;

c) Oligonucleotides #1 and #15 (Table 2) for amplification of the finalproduct, 50 picomoles each in 100 μl final volume.

Promoter was amplified by stepdown PCR, as described for the chimericPrrn promoters above; the annealing temperatures were between 72° C. to57° C.

PrrnLT7g10+DB/pt Promoter in Plasmid pHK19

The promoter fragment was obtained in one PCR step as shown in FIG. 7.The reaction contained:

a) The product of the PCR-2 reaction generating promoterPrrnLT7g10+DB/Ec in plasmid pHK18 as DNA template; and

b) Oligonucleotides #1 and #16 (Table 2), 50 picomoles each in 100 μlfinal volume.

Promoter was amplified by stepdown PCR, as described for theconstruction of chimeric Prrn promoters above; the annealingtemperatures were between 72° C. to 52° C.

PrrnLT7g10−DB Promoter in Plasmid pHK20

The promoter fragment was obtained in one PCR step, which is similar tothe PCR-3 step in FIG. 5. The reaction contained:

a) The product of the PCR-2 reaction generating promoterPrrnLT7g10+DB/Ec in plasmid pHK18 as DNA template; and

b) Oligonucleotides #1 and #14 (Table 2), 50 picomoles each in 100 μlfinal volume.

Promoter was amplified by stepdown PCR, as described for the chimericPrrn promoters above; the annealing temperatures were between 72° C. to52° C.

The final PCR products were digested with the SacI and NheI restrictionenzymes and cloned in plasmid pHK3 to obtain plasmids pHK18, pHK19,pHK20.

Construction of Chimeric neo Genes

Construction of the chimeric promoters was described in the precedingsections. For determining effects on levels of protein accumulation, thepromoters were cloned upstream of a kanamycin-resistance encodingconstruct, consisting of the neo coding region and the 3′-UTR of theplastid rbcL gene. Such constructs are available in plasmids pHK2 andpHK3, which carry the same Prrn(L)rbcL(S)::neo::TrbcL gene as aSacI-HindIII fragment. Plasmid pHK2 is a pUC118 vector derivative; pHK3is a pBSIIKS+ derivative. Plasmid maps with relevant restriction sitesare shown in FIG. 8. DNA sequence of the neo gene in plasmids pHK2 andpHK3 is shown in FIG. 9. Note, that in plasmid pHK2 the neo gene has anEcoRI site upstream of the SacI site (FIG. 8). Prrn and TrbcL have beendescribed by Staub and Maliga, 1994; the neo gene derives from plasmidpSC1 (Chaudhuri and Maliga, 1996). The pUC118 and pBSIIKS+plasmidderivatives which carry the various promoter constructs are listed inTable 1.

To determine the DNA sequence of the promoter fragments, the plasmidswere purified with the QIAGEN Plasmid Purification Kit following themanufacturer's recommendations. DNA sequencing was carried out using aT7 DNA sequencing kit (version 2.0 DNA, Amersham Cat. No. US70770) andprimer No. #23 in Table 2, which is complementary to the neo codingsequence. These promoter sequences are shown in FIG. 3A-D.

Introduction of Chimeric neo Genes into the Tobacco Plastid Genome

Suitable vectors are available for the introduction of foreign genesinto the tobacco plastid genome. Such vectors are pPRV111A and pPRV111B,which carry a selectable spectinomycin-resistance (aadA) gene and targetinsertions into the repeated region of the plastid genome (Zoubenko etal., 1994). The chimeric neo genes were cloned into one of these plastidtransformation vectors (Table 1) and introduced into the tobacco plastidgenome by the biolistic process. From the transformed cells plants wereregenerated by standard protocols (Svab and Maliga, 1993). A uniformpopulation of transformed plastid genome copies was confirmed bySouthern analysis.

For Southern analysis, total cellular DNA was prepared by the CTABmethod (Saghai-Maroof et al., 1984). Two leaves of each transformedplant were homogenized and incubated at 60° C. for 30 minutes in abuffer containing 2% CTAB (tetradecyl-trimethyl-ammonium bromide), 1.4 MNaCl, 20 mM EDTA (pH 8.0), 1 mM Tris/HCl (pH 8.0) and 100 mMβ-mercaptoethanol. After chloroform extraction, the DNA was precipitatedwith isopropyl alcohol and dissolved in water or in TE buffer (10 mMTris, 1 mM EDTA, pH 8.0). DNA digested with an appropriate restrictionenzyme was electrophoresed on 0.8% agarose gel and transferred to nylonmembrane using PosiBlot Transfer apparatus (Stratagene). The blots wereprobed using Rapid Hybridization Buffer and plastid targeting sequencesas a probe labeled with random priming (³²P, Boehringer Mannheim Cat No.1004760).

Plastid transformation was achieved with each of the plasmids listed inTable 1. Exceptions were plasmids pHK41 and pHK42. It appears that NPTIIexpression with the psbA leader derivatives was so high that the plantswere not viable. It follows that these same leaders may be used toadvantage when fused with weaker promoters.

Transplastomic lines are designated by Nt (N. tabacum, the species), theplasmid name (for example pHK30) and an individual line number and aletter identifying regenerated plants. For example, the Nt-pHK30-1D andNt-pHK30-1C plants were both obtained by transformation with plasmidpHK30, are derived from the same transformation event and wereregenerated from the same culture. Nt-pHK30-2 plants are derived from anindependent transformation event. Normally, several transformed linesper construct were obtained. However, data are shown here only for one:Nt-pHK30-1D, Nt-pHK31-1C, Nt-pHK60-5A, Nt-pHK32-2F, Nt-pHK33-2A,Nt-pHK34-9C, Nt-pHK35-4A, Nt-pHK64-3A, Nt-pHK36-1C, Nt-pHK37-2D,Nt-pHK38-2E, Nt-pHK39-3B, Nt-pHK40-12B and Nt-pHK43-1C.

Testing mRNA Accumulation by RNA Gel Blot (Northern) Analysis

RNA gel blot analysis was performed to determine steady-state levels ofchimeric mRNA in the transplastomic lines. Total leaf RNA was preparedfrom the leaves and roots of plants grown in sterile culture accordingto Stiekema et al (1988). RNA (4 μg per lane) was electrophoresed on 1%agarose gel and transferred to nylon membranes using the PosiBlotTransfer apparatus (Stratagene). The blots were probed using RapidHybridization Buffer Amersham) with a ³²P-labeled neo probe (Pharmacia,Ready-To-Go Random Priming Kit). The neo probe was obtained by isolatingthe NheI/XbaI fragment from plasmid pHK2. The template for probing thetobacco cytoplasmic 25S rRNA was a fragment which was PCR amplified fromtotal tobacco cellular DNA with primers 5′-TCACCTGCCGAATCAACTAGC-3′ (SEQID NO: 56) and 5′-GACTTCCCTTGCCTACATTG-3′ (SEQ ID NO: 57). RNAhybridization signals were quantified using a Molecular DynamicsPhosphorImager, and normalized to the 25S rRNA signal.

Testing NPTII Accumulation by Protein Gel Blot (Western) Analysis

Total soluble protein was extracted from the leaves, roots or seeds oftransgenic tobacco plants grown in sterile culture. In case of leavesgrown in sterile culture, about 200 mg leaf tissue was homogenized in 1ml of buffer containing 50 mM Hepes/KOH (pH 7.5), 1 mM EDTA, 10 mMpotassium acetate, 5 mM magnesium acetate, 1 mM dithiothreitol and 2 mMPMSF. The homogenate was centrifuged twice at 4° C. to remove insolublematerial. Protein concentration was determined using the Biorad ProteinAssay reagent kit. Transgenic tobacco plants expressing neo in theplastid genome (Nt-pTNH32-70, Carrer et al., 1993) and wild type plantswere used as positive and negative controls, respectively. Proteins wereseparated in SDS polyacrylamide gels (SDS-PAGE; 15% acrylamide, 6 Murea) and transferred to nitrocellulose membranes using a semi-drytransfer apparatus (Bio-Rad). After blocking non-specific binding sites,the membrane was incubated with 4,000-fold diluted polyclonal rabbitantiserum raised against NPTII (5Prime-3Prime Inc.). HRP-conjugatedsecondary antibody, diluted 20,000 fold, and ECL chemiluminescence wereused for immunoblot detection on X-ray film. NPTII was quantified on theimmunoblots by comparison of the experimental samples with a dilutionseries of commercial NPTII (5Prime-3Prime).

EXAMPLE 1 DB Sequences Enhance Protein Accumulation from rbcL Leader;Protein Accumulation from the atpB Translation Control Signals is Highbut DB-Independent

The role of DB sequences in mRNA translation was tested using neo as thereporter gene. The neo gene encodes the bacterial enzyme neomycinphosphotransferase (NPTII)(Beck et al., 1982). The tested neo genes havethe same promoter (Prrn) and transcription terminator (TrbcL), anddiffer only with respect to the translation control region (TCR)comprising the 5′ untranslated region of the mRNA and the coding regionN-terminus. Two constructs were prepared with the atpB and rbcL TCRs.One construct contained the wild-type TCR, including the processed 5′untranslated region and 42 nucleotides of the coding region N-terminus(PrrnLatpB+DBwt, plasmid pHK30, FIG. 4B; PrrnLrbcL+DBwt, plasmid pHK34,FIG. 4A). The second construct contained silent mutations in the42-nucleotide segment of the atpB and rbcL N-terminal coding regions toeither eliminate or alter mRNA and rRNA base pairing (PrrnLatpB+DBmplasmids pHK60, FIG. 2A and FIG. 4B; PrrnLrbcL+DBm, pHK64, FIG. 2A andFIG. 4A). The silent mutations altered the mRNA sequence withouteffecting the amino acid sequence. For example, 13 potential base pairsmay form between the wild-type atpB mRNA and the ADB sequence shown atthe bottom in FIG. 2A. The 11 silent mutations affect eight base-paringevents for this particular ADB−DB interaction. After mutagenesis, thereis a possibility for ten base pairing events, most of which are new. Thechimeric neo genes were introduced into the tobacco plastid genome byhomologous targeting using the biolistic approach (Svab and Maliga,1993; Zoubenko et al., 1994). NPTII and neo mRNA levels were thenassessed in the leaves of transplastomic plants. Since NPTII inwild-type DB-containing and mutant DB-containing plants has the exactsame protein sequence, protein levels in the plants directly reflect theefficiency of mRNA translation. In case of the atpB TCR, mutagenesis ofDB reduced protein accumulation to ˜4% instead of ˜7% (FIG. 10 and Table3). In contrast, mutagenesis of rbcL DB had a dramatic effect, reducingNPTII accumulation 35-fold. Thus, DB-ADB interaction is very importantfor translation of the plastid rbcL mRNA, but is less important fortranslation of the atpB mRNA.

We also prepared a third construct set with the atpB and rbcL leaders,but without the native DB (PrrnLatpB−DB, plasmid pHK31, FIG. 4B;PrrnLrbcL−DB, plasmid pHK35, FIG. 4A). The neo coding region in theseconstructs is directly linked to the Prrn promoter via a synthetic NheIrestriction site. The NheI restriction site (GCTAGC) is fullycomplementary to the ADB region (FIG. 2B), therefore it was hoped thatit would function as a DB sequence. Utility of NheI site as analternative DB could be best judged by NPTII accumulation from the rbcLleader, which is highly dependent on DB. High levels of NPTII from theNheI construct (4.7%) relative to the mutant DB (0.3%) indicate, thatlinking the coding region via an NheI site provides a suitable DB forexpressing foreign polypeptides (FIG. 10, Table 3). TABLE 3 Levels ofNPTII and neo mRNA in tobacco leaves NPTII/ SD DB NPTII(%) neo mRNA neomRNA Nt-pTNH32-70 + − 2.10 ± 0.33 41.5 5.06 Nt-pHK30-1D (+) wt 7.02 ±0.82 70.05 ± 12.33 8.85 Nt-pHK31-1C (+) s 2.52 ± 0.79 100 2.52Nt-pHK60-5A (+) m 4.03 ± 1.45 91.57 ± 12.76 4.40 Nt-pHK32-2F − wt 1.17 ±0.05 49.33 ± 7.76  2.37 Nt-pHK33-2A − s 0.21 ± 0.05 49.55 ± 6.67  0.42Nt-pHK34-9C + wt 10.83 ± 3.84  48.91 ± 22.65 22.14 Nt-pHK35-4A + s 4.68± 1.84 21.41 ± 7.88  21.86 Nt-pHK64-3A + m 0.31 ± 0.15 52.47 ± 4.29 0.59 Nt-pHK36-1C + wt  2.17 ± 70.97 68.8 3.15 Nt-pHK37-2D + s 2.35 ±0.05 42.3 5.56 Nt-pHK38-2E + Ec 16.39 ± 3.42  47.59 ± 19.06 34.44Nt-pHK39-3B + pt 0.16 ± 0.13 13.12 ± 1.27  1.22 Nt-pHK40-12B + s 23.00 ±5.40  90.27 ± 31.83 25.48 Nt-pHK43-1C (+) s 0.65 ± 0.28 13.2 4.92

Discussion

In bacteria, mutagenesis or deletion of the DB reduces translation 2- to34-fold, depending on the individual mRNA (Etchegaray and Inouye, 1999;Faxen et al., 1991; Ito et al., 1993; Mitta et al., 1997; Sprengart etal., 1996). Furthermore, reliance on the DB increases when the SDsequence is removed (Sprengart et al., 1996; Wu and Janssen, 1996). Inour experiments, no variation was made in the atpB or rbcL 5′UTR, onlysequences downstream of the AUG were altered. Mutagenesis of the atpB DBregion reduced protein levels ˜2-fold. Although the atpB mRNA does nothave a SD directly upstream of AUG, we speculate that it probably has analternate mechanism for translation initiation that reduces itsdependence on the DB. Alternatively translation initiation may befacilitated by activator proteins as described for Chlamydomonaschloroplasts (Rochaix, 1996; Stern et al., 1997). The consequence of DBmutagenesis on rbcL translation was a dramatic 35-fold drop in NPTIIlevels. Accordingly, efficient rbcL translation is highly dependent onDB-ADB interactions. Genes in both prokaryotes and eukaryotes showbiases in the usage of the 61 amino acid codons and have a tRNApopulation closely matched to the overall codon bias of the residentmRNA population. Incorporation of synonymous minor codons in the codingregion may dramatically reduce translation (Makrides, 1996) anddestabilize the mRNA (Deana et al., 1998). A well-characterized examplefor minor codons causing reduced expression in E. coli are the AGA/AGGarginine codons recognized by the same tRNA which are present at thefrequency of 2.6 and 1.6 per thousand codons. Therefore, we havecompared codon usage bias and frequency of triplets per 1000 nucleotidesin the wild-type and mutagenized atpB and rbcL DB regions. Since westudied NPTII accumulation in leaves, the values shown in FIG. 12 werecalculated for the highly expressed rbcL, psaA, psaB, psaC, psbA, psbB,psbC, psbD, psbE and psbF photosynthetic genes using the GeneticsComputer Group (GCG; Madison Wis.) codon frequency program. Codon usagebias and triplet frequency is comparable in the wild-type and mutant DBregions of both atpB and rbcL. In addition, the mRNAs for the wild-typeand mutant DB constructs accumulate at similar levels. Therefore, thedramatic change in NPTII acccumulation from the PrrnLrbcL+DBm promoterin the Nt-pHK64 line can not be attributed to incorporation of a rarecodon in the mutant DB region.

We have shown here that sequences downstream of the translationinitiation codon may dramatically affect mRNA translation. Therefore,silent mutations in the DB region of heterologous proteins maysignificantly improve expression in chloroplasts by increasingcomplementarity of the mRNA with the plastid rRNA penultimate stemstructure.

There are significant differences in NPTII accumulation from neotransgenes with different leaders and the same synthetic DB (Table 3).This indicates that the 5′UTR is an important determinant of translationefficiency. Many data are available supporting the importance of 5′UTRas a target for translational control in higher plants (Hirose andSugiura, 1996; Staub and Maliga, 1993; Staub and Maliga, 1994b) and theunicellular alga Chlamydomonas (Mayfield et al., 1994; Nickelsen et al.,1999; Sakamoto et al., 1993; Zerges et al., 1997). The data presentedherein demonstrate that translation efficiency in plastids is determinedby sequences both upstream and downstream of the AUG.

EXAMPLE 2 Study of Phage T7g10 Translation Control Sequences Indicatesthat the Efficient DB in Plastids has Loose Complementarity to ADB

Since the actual ADB sequence is different in plastids and E. coli, weanticipated (Sprengart et al., 1996; Etchegaray & Inoyue, 1999) thatreplacement of the E. coli DB with a perfect plastid DB (100% DB-ADBcomplementarity) would enhance translation in plastids. We choose thephage T7g10 translational control region for the study since it has awell-characterized E. coli DB. Three Prrn promoter derivatives wereconstructed. Cassette PrrnLT7g10+DB/Ec consists of Prrn fused with thenative T7g10 TCR containing the E. coli DB (plasmid pHK38; FIG. 2B, FIG.4A). Cassette PrrnLT7g10+DB/pt consists of the Prrn promoter, T7g10leader and the perfect tobacco DB (pHK39; FIG. 2B, FIG. 4A). CassettePrrnLT7g10−DB has the Prrn promoter and T7g10 leader, but lacks theT7g10 DB sequence (pHK40; FIG. 2B, FIG. 4A). The neo coding region inthese constructs is directly linked to the Prrn promoter via a syntheticNheI restriction site. The neo genes in the three expression cassetteswere introduced into tobacco plastids by transformation (Svab andMaliga, 1993; Zoubenko et al., 1994) and the leaves of transplastomictobacco were tested for NPTII accumulation and mRNA levels (FIGS. 10,11; Table 3).

Surprisingly, NPTII levels from the heterologous T7g10 TCR were higher(Nt-pHK38; ˜16%) than the levels obtained from the rbcL TCR (Nt-pHK34;˜11%). We expected that incorporation of the plastid DB with 100%complementarity would further enhance NPTII levels. Instead, we foundthat plants transformed with the construct having the perfect plastid DB(Nt-pHK39) contained NPTII levels 100-fold lower than the plantsexpressing NPTII from the E. coli TCR (Nt-pHK38; FIGS. 10; Table 3).This result suggests that, unlike in E. coli, 100% complementarityreduces, rather than enhances translation efficiency. Indeed, none ofthe highly expressed plastid genes have a perfect DB sequence (FIG. 2A).RNA gel blots shown in FIG. 11 indicate that Nt-pHK39 plants with theperfect DB contain ˜3-fold less neo mRNA. Therefore, a contributingfactor to lower NPTII levels in these plants appears to be a faster mRNAturnover rate. Furthermore, NPTII expressed from the PrrnLT7g10derivatives differ by the DB-encoded amino acids at the N-terminus.Therefore, differential protein turnover rates may be part of the reasonfor differences in NPTII accumulation. The highest yield of NPTII (23%)was obtained with the synthetic, NheI-containing DB cassette.

Discussion

This example utilizing the rbcL translation control regions reveals thatsequences downstream of the translation initiation codon maydramatically affect mRNA translation. Therefore, silent mutations in theDB region of heterologous proteins may significantly improve expressionin chloroplasts by increasing complementarity of the mRNA with theplastid rRNA penultimate stem structure. However, it appears thatperfect complementarity is undesirable, as it may accelerate mRNAturnover and reduce the rate of translation. This finding highlightsdifferences in the translation machinery of plastids and E. coli, inwhich perfect complementarity enhances translation (Etchegaray andInouye, 1999; Sprengart et al., 1996). It is possible, however, thatshifting the region of complementarity relative to AUG or targeting aslightly different region of the penultimate stem may facilitate highlyefficient translation of mRNAs with a perfectly matched DB.

The T7g10 constructs have one or two relatively rare AGC serine codons(4.7 per 1000, FIG. 12), one of which is encoded in the NheI site. Thiscodon is present in the Nt-pHK38 and Nt-pHK40 plants, which contain thehighest levels of NPTII. Further improvement may be expected byreplacing the AGC with an AGT serine codon.

EXAMPLE 3 The clpP, psbB and psbA TCRs have Distinct ExpressionCharacteristics

NPTII accumulation was studied in transplastomic tobacco carrying thePrrnLclpP promoter derivatives. The PrrnLclpP+DBwt (Nt-pHK32-2F) andPrrnLclpP−DB (Nt-pHK33-2A) plants accumulate 1.2% and 0.2% NPTII intheir leaves (FIG. 10; Table 3). We have found that over-expression ofclpP 5′-UTR causes a mutant phenotype manifested as pale green leafcolor and slower growth. This phenotype is normalized in older plants.We assume that the primary cause of this mutant phenotype is the lack ofClpP protein, the clpP gene product. This mutant phenotype is absent inplants transformed with other 5′UTRs. Therefore we believe, that themutant phenotype is attributable to competition for a clpP-specificnuclear factor. The clpP gene has two introns. Preliminary RNA gel blotanalysis reveals reduced levels of mature, monocistronic clpP mRNA (˜30%of wild-type) and accumulation of intron I-containing clpP pre-mRNA inthe pale-green leaves. Normalization of phenotype coincides withincrease of translatable monocistronic clpP mRNA to wild type levels.Over-expression of clpP 5′UTR therefore may interfere with splicing ofclpP pre-mRNA.

NPTII accumulation was also studied in transplastomic tobacco carryingthe PrrnLpsbB promoter derivatives. The PrrnL psbB+DBwt (Nt-pHK36-1C)and PrrnL psbB −DB (Nt-pHK37-2D) plants accumulate 2.2% and 2.4% NPTIIin their leaves (FIG. 10; Table 3). Thus, the synthetic DB sequence incase of the psbB TCR efficiently replaces the native DB sequence.Conversely, it may rely on an alternative mechanism for translationinitiation.

The Prrn promoter constructs with the psbA leader were obtained asdescribed. However, we have been able to introduce only one of them,PrrnLpsbA−DB(+GC) into tobacco plastids in line Nt-pHK43-1C. TheNt-pHK43-1C plants accumulate NPTII at a relatively low level (0.6%;FIG. 10, Table 3). It is conceivable that the lack of success inintroducing the +DB construct is due to the dramatically elevatedexpression level of NPTII which is toxic to the plants.

Discussion

NPTII levels obtained from PrrnLclpP+DBwt (Nt-pHK32-2F) promoter arerelatively low, only 1.2% of the total soluble protein. However, thispromoter is desirable for driving expression of selectable marker genes,as the recovery of transplastomic clones is relatively efficient whenthe neo gene is expressed from this promoter, as shown in Example 4.Expression of neo from the PrrnLclpP+DBwt promoter does not cause amutant phenotype in tissue culture. Thus, it is suitable to drive theexpression of marker genes, so long as the marker gene is subsequentlyremoved. It appears that competition for a nuclear-encoded factorrequired for processing the clpP introns gives rise to the reducedexpression observed. This intron is absent in the clpP genes in themonocots rice (Hiratsuka et al., 1989) and maize (Maier et al., 1995).The PrrnLclpP+DBwt promoter therefore may be used to advantage in thetransformation of monocots. Furthermore, the level of the trans-factorrequired for clpP intron processing is likely to be expressed atdifferent levels in dicots. We anticipate therefore, that expression ofthe clpP TCR will have no undesirable consequences in other dicotspecies. It is also possible that the phenotypic consequences ofexpressing the clpP TCR in plastids is a property of the tobacco line,N. tabacum cv. Petit Havana utilized herein and is absent in othertobacco lines. This would make the clpP gene TCR a desirable expressiontool in both monocots and dicots.

Both psbB leader derivatives accumulate NPTII at comparable levels (2.2%and 2.4%, respectively; Table 3). This 5′ regulatory region is a goodalternative to the most commonly used rbcL leader when proteinaccumulation is required in the ˜2% range.

In the past, the psbA promoter and leader construct yielded relativelyhigh levels of expression in leaves (2.5% GUS; Staub and Maliga, 1993).Yet these constructs did not contain psbA DB elements. The presentinvention describes the generation of chimeric promoters that aresuitable to obtain high-level protein expression while elucidating theregulatory role played by DB sequences. Prrn is the strongest knownpromoter in plastids and consequently provides for high levels of NPTIItranslation. These elevated levels of NPTII can be toxic to the plantand therefore it is difficult to obtain transplastomic lines with thehighest prospective levels of NPTII. An alternative approach involvesoperably linking the psbA leader to a relatively weak promoter. Thisapproach may generate cassettes which are suitable for obtainingrelatively high levels of protein accumulation from relatively lowlevels of mRNA.

EXAMPLE 4 NPTII Accumulation in Roots and Seeds

Posttranscriptional regulation is an important mechanism of plastid geneexpression (Rochaix, 1996; Stem et al., 1997). Therefore, we expectedthat NPTII accumulation may be tissue-specific due to regulation of geneexpression at the level of mRNA translation. Thus, NPTII accumulationwas tested in roots and seeds.

Testing of NPTII accumulation in roots was carried out with a subset oftransplastomic lines (Table 4). Roots for protein extraction werecollected from plants grown in liquid MS salt medium (3% sucrose) insterile cultures incubated on a shaker to facilitate aeration. Proteinwas extracted from the roots with the leaf protocol and tested for NPTIIaccumulation (FIG. 13 A). The highest level of NPTII, 0.75%, is found inthe roots of plants expressing NPTII from the clpP TCR (PrrnLclpP+DBwtconstruct; pHK32). The second highest value, 0.3%, was found in theroots of plants transformed with plasmid pHK38 expressing NPTII from theT7g10 TCR (PrnnLT7g10+DB/Ec promoter). The level of NPTII was about thesame, approximately 0.1%, in roots expressing the recombinant proteinfrom the atpB and rbcL TCR in pHK30- and pHK34-transformed plants.

Since plastids in the roots are smaller than in leaves, we expectedlower levels of NPTII accumulation in the roots than in the leaves. Thiswas true for all the tested roots, except those of the Nt-pHK32 plants.Interestingly, NPTII from the clpP TCR accumulated at almost the samelevel in the roots (0.75%, Table 4) as in the leaves (approximately 1%,Table 3). This is likely attributable to high levels of the neo mRNA inthe roots (FIG. 13B). Since the clpP leader includes the minimalPclpP-53 promoter (Sriraman et al., 1998a; NAR 26: 4874) we speculate,that the relatively high mRNA levels are due to activation of PclpP-53in roots. High levels of expression make the clpP leader a desirable TCRfor protein expression in roots.

The T7g10 leader (pHK38) was the most efficient in roots from which themost NPTII accumulated relative to the mRNA (Table 4). Although in theNt-pHK38 plants, the neo mRNA was 7-times less than in the Nt-pHK32plants, NPTII levels were almost as high (approximately 0.30% comparedto 0.75%) as in the plastids with the clpP TCR (pHK32). High level NPTIIaccumulation from the T7g10 TCR in leaves (pHK38, pHK40; Table 3) and inroots (pHK38; Table 4) indicates the general utility of the phage T7g10translation control region for protein expression in plastids.

Protein accumulation was also studied in seeds harvested from thetransgenic plants (FIG. 14). Protein levels were 0.05% in plantstransformed with pHK32 (clpP TCR), and approximately 0.01% in plantstransformed with plasmid pHK30 (atpB TCR). No NPTII was detectable inplants in which neo was introduced in the rbcL TCR-construct (plasmidpHK34), indicating differential protein accumulation which is dependenton the choice of the TCR. TABLE 4 Levels of NPTII and neo mRNA intobacco roots Strain NPTII (%) neo mRNA (%) NPTII/neo mRNA × 10³Nt-pHK30-1D 0.14 ± 0.05 33.7 4.2 Nt-pHK32-2F 0.75 ± 0.35 100 7.5Nt-pHK34-9C 0.12 ± 0.03 23.5 5.1 Nt-pHK38-2E 0.31 ± 0.04 13.4 23.1

EXAMPLE 5 High-Level NPTII Expression Facilitates Efficient Recovery ofTransplastomic Lines by Selection for Kanamycin Resistance

The plastid genome of higher plants is a 120-kb to 160-kbdouble-stranded DNA which is present in 1,900 to 50,000 copies per leafcell (Bendich, 1987). To obtain genetically stable transplastomic linesevery one of the plastid genome copies (ptDNA) should be uniformlyaltered in a plant. Since integration of foreign DNA always occurs byhomologous recombination, plastid transformation vectors containsegments of the plastid genome to target insertions at specificlocations. Useful, non-selectable genes are cloned next to theselectable marker genes, which are then introduced into the plastidgenome by linkage to the selectable marker gene (Maliga, 1993).Transforming DNA is introduced into plastids by the biolistic process(Svab et al., 1990; Svab and Maliga, 1993) or PEG treatment (Golds etal., 1993; O'Neil et al., 1993). Elimination of wild-type genome copiesoccurs during repeated cell divisions on a selective medium. The successof transformation depends on the success of selective amplification ofthe few initially transformed genome copies. Therefore the choice of theantibiotic used for the selective amplification of transformed genomecopies and the mechanism by which the plant cells are protected fromantibiotic action is a critical parameter to be considered forsuccessful generation of homoplasmic plants.

The most commonly used antibiotic for the selection of transplastomiclines is spectinomycin, an inhibitor of protein synthesis on plastidribosomes. Initially, plastid transformation in tobacco was carried outby selection for resistance based on mutations in the plastid 16S rRNA(Svab et al., 1990). Selection was inefficient, yielding about onetransplastomic clone per 50 bombarded samples, probably because the 16SrRNA based mutation in recessive. Recovery of transplastomic lines wasenhanced ˜100-fold by selection for a dominant marker, spectinomycinresistance based on inactivation by aminoglycoside 3″ adenyltransferaseencoded in a chimeric aadA gene (Svab and Maliga, 1993). In addition totobacco, selection for spectinomycin resistance (aadA) could be appliedto recover transplastomic lines in Arabidopsis and potato. The aadA genein plants confers resistance to both spectinomycin and streptomycin.Selection for streptomycin resistance was used for plastidtransformation in rice, a species resistant to spectinomycin, afterbombardment with a chimeric aadA gene. See Example 8.

The need for an alternative marker gene for plastid manipulation has ledto testing kanamycin resistance as a selective marker. A chimeric neo(kan) gene, encoding neomycin phosphotransferase, was suitable torecover transplastomic tobacco lines. However, recovery oftransplastomic lines was relatively inefficient, yielding only onetransplastomic line in ˜25 bombarded leaf samples. Furthermore, forevery plastid transformation event ˜25 to 50 kanamycin resistant lineswere obtained in which integration of the plastid neo construct into thenuclear genome resulted in kanamycin resistance (Carrer et al., 1993).We report here that the efficiency of recovering transplastomic clonesis significantly improved when transforming tobacco chloroplasts with anew neo gene expressed from a promoter with the atpB and clpPtranslation control region. The number of nuclear transformation eventsis reduced using the cassettes of the present invention. Theseimprovements make the new neo gene a practical tool for plastid genomemanipulations.

Discussion

The chimeric neo genes described in Examples 1-4 were introduced intoplastids by selection for the linked spectinomycin resistance (aadA)gene as their suitability for directly selecting transplastomic lineswas unknown. The transplastomic lines listed in Table 3 were then testedfor resistance to kanamycin by their ability to proliferate on a mediumcontaining 50 mg/L kanamycin. The RMOP meduim used for testing inducesformation of green callus and shoot regeneration in the absence ofkanamycin. The tissue culture procedures utilized for this example aredescribed in references Carrer et al., 1993 and Carrer and Maliga, 1995.

On the selctive kanamycin medium only scanty, white callus forms fromwild-type leaf section. Formation of green callus and shoots from leafsection of plants transformed with pHK plasmids in Table 3 indicatesthat accumulation of NPTII confers kanamycin resistance. We set out totest if transplastomic clones can be directly selected by kanamycinresistance after bombardment with plasmids pHK30 and pHK32. The resultsare summarized in Table 5.

Bombardment of 25 tobacco leaves with plasmid pHK30 yielded 45 kanamycinresistant lines on a medium containing 50 mg/L kanamycin. Transplastomicneo lines are expected to be resistant to much higher levels, 500 mg/Lof kanamycin (Carrer et al., 1993). In addition, in plasmid pHK30 theneo gene is physically linked to a spectinomycin resistance (aadA) gene.Spectinomycin resistance is manifested as kanamycin resistance:sensitive leaf sections form white callus and no shoots whereasresistant leaf sections form green callus and shoots on a selectivemedium (500 mg/L) RMOP medium. We assumed therefore, that alltransplastomic lines should be resistant to both 500 mg/L of kanamycinand 500 mg/L spectinomycin (Carrer and Maliga, 1995). When applying thistest we found that 22 of the 45 lines meet these criteria. Digestion ofthe plastid DNA with the EcoRI restriction enzyme and probing with theplastid targeting region should detect 3.1-kb fragment in the wild-typeand a 4.2-kb and 1.2-kb fragment in transplastomic lines (FIG. 15A). DNAgel blot analysis of seven of the kanamycin-spectinomycin resistantlines confirmed integration of both transgenes into the plastid genome(FIG. 15B). Therefore, we assume that all 22 kanamycin-spectinomycinlines are transplastomic (Table 5).

Bombardment of 30 tobacco leaves with plasmid pHK32 yielded 28 kanamycinresistant lines on a medium containing 50 mg/L kanamycin. We haveidentified 11 double-resistant lines by testing these on a mediumcontaining 500 mg/L of kanamycin and 500 mg/L spectinomycin. All sixtested were transplastomic by DNA gel blot analysis (FIG. 15B),therefore we believe that all eleven are transplastomic (Table 5). TABLE5 SELECTION OF TRANSPLASTOMIC TOBACCO CLONES BY KANAMYCIN RESISTANCEKan. Res. 500 mg/L No. Kan. Res. Kan. Res. Spec. Res. Vector leaves 50mg/L 500 mg/L 500 mg/L Transplastomic pTNH32 29 59 7 0 50^(a) 52 225^(a) 47 4 1 pHK30 25 45 22 22 pHK32 30 28 11 11(^(a)Carrer et al., 1993)

Discussion

Plastid transformation efficiency should be comparable, if we target thesame region of the plastid genome for insertion, use similar sizetargeting sequences and the same method of DNA delivery. Therefore,lower transformation efficiencies obtained by selection for kanamycinresistance with the old chimeric neo genes was likely due to the lack ofrecovery of tranplastomic clones by selection. We have found thattransformation with neo genes expressed from the PrrnLatpB+DBwt andPrrnLclpP+DBwt promoters is as efficient as with the aadA gene. This isa significant technical advance, and will facilitate plastidtransformation in crops, in which the regenerable tissues containnon-green plastids. Most important targets are the non-green plastids ofcereal crops. Kanamycin selection is widely used to obtain transgeniclines after transformation with chimeric neo genes in dicots. However,kanamycin is an undesirable selective agent in monocots such as cerealtissue cultures. However, NPTII also inactivates paromomycin, which maybe used to recover nuclear gene transformants at an extremely highefficiency in cereals. See for example, PCT application WO99/05296.

EXAMPLE 6 Bacterial Bar Gene Expression in Tobacco Plastids ConfersResistance to the Herbicide Phosphinothricin

Bialaphos, a non-selective herbicide, is a tripeptide composed of twoL-alanine residues and an analog of glutamic acid known asphosphinothricin (PPT). While PPT is an inhibitor of glutaminesynthetase in both plants and bacteria, the intact tripeptide has littleor no inhibitory effect in vitro. Bialaphos is toxic for bacteria andplants, as intracellular peptidases remove the alanine residues andrelease active PPT. Bialaphos is produced by Streptomyces hygroscopicus.The bacterium is protected from phosphinothricin toxicity byphosphinothricin acetyltransferase (PAT), the bar gene product. Thisenzyme acetylates phosphinothricin or demethylphosphinothricin (Thompsonet al., 1987). PPT resistant crops have been obtained by expressing theS. hygroscopicus bar gene in the plant nucleus. Herbicide resistantlines were obtained by direct selection for PPT resistance in cultureafter Agrobacterium tumefaciens-mediated DNA delivery in tobacco,potato, Brassica napus and Brassica oleracea (De Block et al., 1987,1989). Biolistic DNA delivery of chimeric bar genes has been employed toobtain PPT resistant maize (Spencer et al., 1990), rice (Cao, et al,1992) and Arabidopsis thaliana (Sawaskaki et al., 1994). Construction oftransplastomic tobacco plants, in which PPT resistance is based on theexpression of bar from S. hygroscopicus in plastids is described in thepresent example. The vectors utilized to express the bar gene contain anexemplary chimeric 5′ regulatory region as set forth in the previousexamples. The following material and methods facilitate the practice ofthis aspect of the present invention.

Construction of Plastid Bar Gene

A NcoI/XbaI bar gene fragment was generated by PCR amplification usingplasmid of pDM302 (Cao et al., 1992) with the following primers: P1,5′-AAACCATGGCACCACAAACAGAGAGCCCA (SEQ ID NO:58) GAACGACGCCC-3′; P2,5′-AAAATCTAGATCATCAGATCTCGGTGACG-3′. (SEQ ID NO:59)

The ends of the PCR fragment were blunt ended by treatment with theKlenow Fragment of DNA polymerase I. The fragment was then ligated intothe EcoRV site of pBluescript II KS+ (Stratagene, La Jolla, Calif.) tocreate plasmid pJEK3. Sequence analysis of pJEK3 plasmid DNA revealedthat the XbaI site we intended to create through PCR amplification ofpDM302 is absent. See FIG. 19. The bar gene has the two translationtermination codons followed by vector sequences. The last 20 bp of pJEK3are: CCCGTCACCGAGATCTGATGAtcgaattcctgcagcccgggggatccactagttct aga (SEQID NO: 133). The bar sequences are in capital (stop codons underlined),the vector sequences are in lower case (XbaI site underlined). Sincethere is an XbaI site present in the vector 40 bp from the intended XbaIsite, it was not necessary to repair this error. The NcoI-XbaI fragmentfrom plasmid pJEK3 was ligated into NcoI-XbaI digested pGS104 plasmid(Serino and Maliga, 1997) to generate plasmid pJEK6. Plasmid pGS104carries a Prrn-TrbcL expression cassette in a pPRV111B plastidtransformation vector. A map of the plastid targeting region of plasmidpJEK6 is shown in FIG. 16A.

Plastid Transformation and Plant Regeneration

Tobacco (Nicotiana tabacum cv. Petit Havana) plants were grownaseptically on agar-solidified medium containing MS salts (Murashige andSkoog, 1962) and sucrose (30 g/l). Leaves were placed abaxial side up onRMOP media for bombardment. The RMOP medium consists of MS salts,N6-benzyladenine (1 mg/l), 1-naphthaleneacetic acid (0.1 mg/l), thymine(1 mg/l), inositol (100 mg/l), agar (6 g/l), pH 5.8, and sucrose (30g/l). The DNA was introduced into chloroplasts on the surface of 1 μmtungsten particles using the DuPont PDS1000He Biolistic gun (Maliga1995). Spectinomycin resistant clones were selected on RMOP mediumcontaining 500 μg/ml spectinomycin dihydrochloride. Resistant shootswere regenerated on the same selective medium and rooted on MS agarmedium (Svab and Maliga, 1993). The independently transformed lines aredesignated by the transforming plasmid (pJEK6) and a serial number, forexample pJEK6-2, pJEK6-5. Plants regenerated from the same transformedline are distinguished by letters, for example pJEK6-2A, pJEK6-2B.

Southern Blot Analysis

Total cellular DNA was isolated from wild-type and transgenicspectinomycin resistant plants with CTAB (Saghai-Maroof et al., 1984).The DNA was digested with the Sma I and BglII restriction endonucleases,separated on a 0.7% agarose gel and blotted onto a Hybond-N nylonmembrane (Amersham, Arlington Heights, Ill.) by a pressure blotter. Themembrane was hybridized overnight with an ApaI/BamHI fragment labeledwith (α-³²P)dCTP using a dCTP DNA Labeling Beads Kit (Pharmacia Inc,Piscataway, N.J.). The membrane was washed 2 times with 0.1×SSPE,0.2×SDS at 55° C. for 30 minutes. Film was exposed to the membrane for30 minutes at room temperature.

PAT Assay

The PAT assay was performed as described by Spencer et. al. (1990). Leaftissue (100 mg) from wild type tobacco (wt), transgenic Nt-pDM307-10tobacco (a line transformed with the nuclear bar gene in plasmid pDM307;Cao et al., 1992), and plastid bar gene transformants was homogenized in1 volume of extraction buffer (10 mM Na₂HPO₄, 10 mM NaCl). Thesupernatant was collected after spinning in a microfuge for 10 minutes.Protein (25 mg) was added to 1 mg/ml PPT and ¹⁴C-labeled Acetyl CoA. Thereaction was incubated at 37° C. for 30 minutes and the entire reactionwas spotted onto a TLC plate. Ascending chromatography was performed ina 3:2 mixture of 1-propanol and NH₄OH. Film was exposed to the TLC plateovernight at room temperature.

Herbicide Application

Wild type and transgenic plants were sprayed with 5 ml of a 2% solutionof Liberty (AgrEvo, Wilmington, Del.) with an aerosol sprayer.

Results and Discussion

First the bacterial bar gene was converted into a plastid gene bycloning the bar coding region into a plastid expression cassette. Thiscassette consists of an engineered plastid rRNA operon promoter (Prrn)and TrbcL and the 3′ UTR of the plastid rbcL gene for stabilization ofthe mRNA. The plastid bar gene was then cloned into the plastidtransformation vector to yield plasmid pJEK6, and introduced intoplastids on the surface of microscopic tungsten particles. The bar geneintegrated into the plastid genome by two homologous recombinationevents via the plastid targeting sequences, as shown in FIG. 16A.Selection for the linked aadA (spectinomycin resistance) gene onspectinomycin-containing medium eventually yielded cells which carried auniformly transformed plastid genome population, which were thenregenerated into plants.

Integration of bar and aadA was verified by DNA gel blot analysis. Totalcellular DNA of wild-type and transplastomic plants was digested withthe SmaI and BglII restriction enzymes and probed with the 2.9-kbApaI-BamHI plastid targeting fragment of N. tabacum (FIG. 16B). The twofragments that were expected for the transgenic plants, 3.3 kb and 1.9kb, were present in each of the transplastomic samples shown in FIG.16B. Absence of the 2.9 kb wild type fragment indicated, that by thetime these plants have been regenerated, the wild-type plastid genomecopies have been diluted out on the selective medium.

To determine if the plastid bar gene has been expressed, leaf extractswere assayed for phosphinothricin acetyltransferase (PAT) activity.Conversion of PPT into acetyl-PPT indicated PAT activity in each of thetested transplastomic lines. Data in FIG. 17 are shown for thetransplastomic lines Nt-pJEK6-2D, Nt-pJEK6-5A and Nt-pJEK6-13B.Interestingly, PAT activity was significantly (>>10-fold) higher whenbar was expressed in the plastids, as compared to the bar gene expressedfrom the cauliflower mosaic virus ³⁵S promoter in the nucleus of theNt-pDM307-10 plant.

PAT expression confers resistance to PPT in tissue culture and in thegreenhouse. When wild type leaf sections are grown in tissue culture, 10mg/L PPT completely blocks callus proliferation. This same PPTconcentration is suitable for the selection of nuclear transformantsafter bombardment with the nuclear bar construct in plasmid pDM307. Leafsections of plants expressing bar in plastids show resistance in thepresence of up to 100 mg/L PPT in the culture medium. We have tested PPTresistance in the greenhouse, spraying wild-type and transplastomicplants with Liberty, a commercial formulation of PPT, at the recommendedfield dose of 2%. As shown in FIG. 18A, 13 days after the treatment, thewild type plants were dead while the transgenic plants thrived. Sincethen the sprayed plants have flowered and set seed. FIG. 18B showsmaternal inheritance of PPT resistance. Lack of plastid pollentransmission results in a lack of herbicide resistance in progenypollinated with transgenic pollen. The bacterial bar gene has a high G+Ccontent (68.3%; Genbank Accession No. X17220), while plastid genes havea relatively high A+T content; for example the G+C content of the highlyexpressed psbA and rbcL genes is 42.7% and 43.7%, respectively (GenbankAccession No. Z00044). Differences in the G+C content are also reflectedin the codon usage biases. Interestingly, data presented here indicatethat expression of bar from S. hygroscopicus is sufficiently high toconfer resistance to field levels of the non-selective herbicide PPT.Furthermore, the PAT enzyme levels obtained in the transplastomic linesare significantly higher than those observed in the nucleartransformant. Therefore, further improvement of the expression levelsmay be obtained by optimizing the codon usage for plastids as set forthin Example 7.

Advantages of incorporating bar in the plastid genome are containment ofherbicide resistance due to the lack of pollen transmission in mostcrops. Furthermore, the lack of genetic segregation would simplifyback-crossing for the introduction of herbicide resistance intoadditional breeding lines.

EXAMPLE 7 A Synthetic Bar Gene Improves Containment and EnhancesExpression in Plastids

The bacterial bar gene was introduced into the tobacco plastid genome bytransformation with plasmid pJEK6, as described above in Example 6. Inplasmid pJEK6 bar is expressed in a cassette consisting of thePrrn(L)rbcL(S) promoter and TrbcL transcription terminator. This plasmidconferred PPT resistance to plants grown in the presence of PPT in thetissue culture medium, but direct selection for transformed lines wasnot possible. Although the PAT levels in homoplastomic leaves was high,the amount of PAT produced by the few pJEK6 bar copies during the earlystage of plastid transformation was probably insufficient to protect theentire cell.

To improve bar expression in plastids a synthetic gene was created. Thecodon usage was modified to mimic that of the average tobaccophotosynthetic plastid gene. Changing the codon usage lead to a loweredGC content characteristic of higher plant plastid genes. To assist withcloning, restriction enzyme recognition sequences were removed and addedas necessary. Codon usage frequency in bacteria reflects relative tRNAabundance: frequent use of codons for rare tRNAs may significantlyreduce translation efficiency. We hoped that differential codon usage inplastids and bacteria would reduce or prevent expression of thesynthetic gene in bacteria, thereby reducing the danger of horizontalgene transfer to microorganisms. We also hoped that improved barexpression in our novel promoter cassettes will allow direct selectionof plastid transformants on PPT-containing medium.

Materials and Methods for Example 7

Codon comparisons of photosynthetic (rbcL, psaA, psaB, psaC, psbA, psbB,psbC, psbD, psbE, psbF) plastid genes were compiled using GCG (GeneticsComputer Group, Madison, Wis.). DNA mutations were then introduced intothe bacterial bar gene making its codon usage more similar to plastidgenes, while removing several restriction enzyme sites that couldinterfere with cloning. See FIG. 28. The synthetic bar gene (s-bar) wasobtained by single-step assembly of the entire s-bar gene from 28oligonucleotides (one 44 nt primer, one 30 nt primer and twenty-six 40nt primers) using PCR (Stemmer et al., 1995). The top and bottom strandsof the primers overlap with each other by 20 nucleotides. NcoI and NheIsites were added at the 5′ end and a XbaI site was added at the 3′ endthrough PCR amplification. To obtain the complete s-bar gene, a smallaliquot of the assembly PCR product was amplified using primers 1A and14B. Unchanged nucleotides are in upper case, altered nucleotides are inlower case in the primers listed below. Primer 1AccATGgctAGCCCAGAAaGAaGaCCG (SEQ ID NO:60) GCCGAtATtaGaCG Primer 1BGCATaTCaGCtTCtGTaGCACGtCta (SEQ ID NO:61) ATaTCGGCCGGtCt Primer 2ATGCtACaGAaGCtGAtATGCCaGCaG (SEQ ID NO:62) TtTGtACaATCGTt Primer 2BCTTGTtTCtATaTAaTGGTTaACGAT (SEQ ID NO:63) tGTaCAaACtGCtG Primer 3AAACCAtTAtATaGAaACAAGtACaGT (SEQ ID NO:64) aAACTTtaGaACtG Primer 3BtTCtTGaGGTTCtTGaGGtTCaGTtC (SEQ ID NO:65) taAAGTTtACtGTa Primer 4AAaCCtCAaGAACCtCAaGAaTGGACt (SEQ ID NO:66) GAtGAtCTaGTCCG Primer 4BAaGGATAGCGCTCtCGtAGACGGACt (SEQ ID NO:67) AGaTCaTCaGTCCA Primer 5ATCTaCGaGAGCGCTATCCtTGGCTtG (SEQ ID NO:68) TaGCaGAaGTtGAC Primer 5BGCGATaCCaGCtACtTCaCCGTCaAC (SEQ ID NO:69) tTCtGCtACaAGCC Primer 6AGGtGAaGTaGCtGGtATCGCaTAtGC (SEQ ID NO:70) GGGCCCtTGGAAGG Primer 6BCCAaTCaTAtGCaTTtCtTGCCTTCC (SEQ ID NO:71) AaGGGCCCGCaTAt Primer 7ACAaGaAAtGCaTAtGAtTGGACaGCt (SEQ ID NO:72) GAaTCaACtGTtTA Primer 7BGtTGaTGaCGtGGtGAaACGTAaACa (SEQ ID NO:73) GTtGAtTCaGCtGT Primer 8ACGTtTCaCCaCGtCAtCAaCGtACaG (SEQ ID NO:74) GACTtGGtTCtACt Primer 8BTTCAGtAGaTGtGTaTAtAGaGTaGA (SEQ ID NO:75) aCCaAGtCCtGTaC Primer 9ACTaTAtACaCAtCTaCTGAAaTCttT (SEQ ID NO:76) GGAGGCACAaGGtT Primer 9BaACAGCtACaACaCTCTTaAAaCCtT (SEQ ID NO:77) GTGCCTCCAaaGAt Primer 10ATtAAGAGtGTtGTaGCTGTtATaGGa (SEQ ID NO:78) tTGCCtAAtGAtCC Primer 10BCtTCaTGCATGCGtACaCtTGGaTCa (SEQ ID NO:79) TTaGGCAatCCtAT Primer 11AaAGtGTaCGCATGCAtGAaGCtCTaG (SEQ ID NO:80) GATATGCtCCaaGa Primer 11BCCtGCaGCCCtCAaCATaCCtCttGG (SEQ ID NO:81) aGCATATCCtAGaG Primer 12AGGtATGtTGaGGGCtGCaGGtTTCAA (SEQ ID NO:82) aCAtGGaAACTGGC Primer 12BtTGCCAaAAACCtACaTCATGCCAGT (SEQ ID NO:83) TtCCaTGtTTGAAa Primer 13AATGAtGTaGGTTTtTGGCAaCTtGAt (SEQ ID NO:84) TTCAGtCTaCCaGT Primer 13BGtAGaACtGGACGaGGaGGTACtGGt (SEQ ID NO:85) AGaCTGAAaTCaAG Primer 14AACCtCCtCGTCCaGTtCTaCCaGTtA (SEQ ID NO:86) CtGAGATCTGATGA Primer 14BtctagaTCATCAGATCTCaGTaACtG (SEQ ID NO:87)

The amplified s-bar coding region was then cloned into a pBSIIKS+plasmid(Stratagene, La Jolla, Calif.) and sequenced (FIG. 20A). The s-bar genewas cloned into cassettes with the chimeric PrrnLatpB+DBwt,PrrnLrbcL+DBwt and PrrnLT7g10+DB/Ec promoters. Table 6 sets forth theplasmids used in the practice of this example.

To provide a suitable cloning site at 3′-end of the bacterial bar gene,the EagI/BglII fragment of s-bar was replaced with the cognate fragmentof the bacterial bar coding region. Such a bacterial bar gene isincorporated in plasmid pKO12 (FIG. 21). In plasmid pKO12 the first 22nucleotides of the bacterial bar coding region are replaced withnucleotides from the s-bar.

Results

The engineered bacterial bar gene in pJEK6 is expressed both in E. coliand plants, as shown in the previous example. We were interested to testif modification of the codon affects expression of the s-bar gene inplastids and in E. coli. In E. coli, s-bar expression was determined bymeasuring PAT activity. Extracts were prepared from bacteria carryingplasmids pKO3 and pKO8 expressing s-bar from the PrrnLatpB+DBwt andPrrnLrbcL+DBwt promoters, respectively. The radioactive assay did notdetect any activity, although extracts from bacteria transformed withplasmids pJEK6 and pKO12 carrying the bacterial bar genes gave strongsignals (FIG. 22A). In plasmid pKO12 the first 22 nucleotides of thebacterial bar coding region are replaced with nucleotides from thes-bar. Therefore, lack of expression from the s-bar in E. coli is notdue to changes within the first 22 nucleotides.

The s-bar was also introduced into plastids by transformation withvector pKO3. Extracts were prepared from pKO3- and pJEK6-transformedtobacco plants, which carry the s-bar and bar genes, respectively.Extracts from both types of plants contained significant PAT activity(FIG. 22B). Therefore, the synthetic bar is expressed in plastids butnot in E. coli.

Changing the bar gene codon usage abrogated expression of the gene in E.coli. This is likely due to the introduction of the rare AGA and AGGarginine codons in the s-bar coding region. The triplet frequency perthousand nucleotides for AGA and AGG is the lowest in E. coli,reflecting low abundance of the tRNA required for translation of thesecodons. The minor arginine tRNA^(Arg(AGG/AGA)) has been shown to be alimiting factor in the bacterial expression of several mammalian genes.The coexpression of ArgU (dnaY) gene that encodes fortRNA^(Arg(AGG/AGA)) resulted in high level production of the targetprotein (Makrides 1996). The bacterial bar gene has 14 arginine codons,none of which are the rare AGA/AGG codons. The s-bar gene has five ofthem, three of which are located within the first 25 codons. Therefore,the likely explanation for the lack of s-bar expression in E. coli isintroduction of the rare AGA and AGG arginine codons in the s-bar codingregion.

There are proteins, which are toxic to E. coli but their expression isdesirable in plastid to which it is not toxic. Engineering of theseproteins in E. coli poses a problem, since the commonly used PEP plastidpromoters are active in E. coli, thus the gene will be transcribed andthe mRNA translated. Incorporation of minor codons in the coding regionwill prevent translation of these proteins in E. coli. Particularlyuseful in this regard is conversion of arginine codons to AGA/AGG. If noarginine is present in the N-terminal region, an N-terminal fusion maybe designed containing multiple AGA/AGG codons to prevent translation ofthe mRNA.

Plants under field conditions are associated with microbes living in thesoil, on the leaves and inside the plants. Gene flow from plastids tothese microorganisms has not been shown. However, it would be an addedsafety measure to incorporate codons in plastid genes, which are rare inthe target microorganisms, but are efficiently translated in plastids.Incorporation of AGA/AGG codons into the selective marker genes and thegenes of interest will prevent transfer of genes from plants tomicrobes, which lack the capacity to efficiently translate the AGA/AGGcodons. In case of specific plant-microbe associations, based ondifferences in codon usage preferences genes could be designed whichwould be expressed in plastids but not in microbes.

Attempts to directly select transplastomic clones after bombardment withthe s-bar constructs so far has failed. The s-bar coding region in FIG.20A contains frequent and rare codons in proportions characteristic ofplastid genes. It is possible, that relatively rare codons in a specificcontext at a critical stage will prevent recovery of plastidtransformation events. Examples for tissue-specific translation of mRNAsdependent on tRNA availability are known (Zhou et al., 1999). Therefore,we designed a second synthetic bar gene, S2-bar, containing onlyfrequent codons (FIG. 20B). Plastid transformation with the s2-bar willenable direct selection of plastid transformation events by PPTresistance.

EXAMPLE 8

Fluorescent Antibiotic Resistance Marker for Facile Identification ofTransplastomic Clones in Tobacco and Rice

Plastid transformation in higher plants is accomplished through agradual process, during which all the 300-10,000 plastid genome copiesare uniformly altered. Antibiotic resistance genes incorporated in theplastid genome facilitate maintenance of transplastomes during thisprocess. Given the high number of plastid genome copies in a cell,transformation unavoidably yields chimeric tissues, in which thetransplastomic cells need to be identified and regenerated into plants.In chimeric tissue, antibiotic resistance is not cell autonomous:transplastomic and wild-type sectors both are green due to phenotypicmasking by the transgenic cells. Novel genes encoding FLARE-S, afluorescent antibiotic resistance enzyme conferring resistance tospectinomycin and streptomycin, which were obtained by translationallyfusing aminoglycoside 3″-adenylyltransferase [AAD] with the Aequoreavictoria green fluorescent protein (GFP) are provided in the presentexample. FLARE-S facilitates distinction of transplastomic and wild-typesectors in the chimeric tissue, thereby significantly reducing the timeand effort required to obtain genetically stable transplastomic lines.The utility of FLARE-S to select for plastid transformation events wasshown by tracking segregation of transplastomic and wild-type plastidsin tobacco and rice plants after transformation with FLARE-S plastidvectors and selection for resistance to spectinomycin and streptomycin,respectively.

Plastid transformation vectors contain a selectable marker gene andpassenger gene(s) flanked by homologous plastid targeting sequences(Zoubenko et al., 1994), and are introduced into plastids by biolisticDNA delivery (Svab et al., 1990; Svab and Maliga, 1993) or PEG treatment(Golds et al., 1993; Koop et al., 1996; O'Neill et al., 1993). Theselectable marker genes may encode resistance to spectinomycin,streptomycin or kanamycin. Resistance to the drugs is conferred by theexpression of chimeric aadA (Svab and Maliga, 1993) and neo (kan)(Carrer et al., 1993) genes in plastids. These drugs inhibit chlorophyllaccumulation and shoot formation on plant regeneration media. Thetransplastomic lines are identified by the ability to form green shootson bleached wild-type leaf sections. Obtaining a genetically stabletransplastomic line involves cultivation of the cells on a selectivemedium, during which the cells divide at least 16 to 17 times (Moll etal., 1990). During this time wild type and transformed plastids andplastid genome copies gradually sort out. The extended period of genomeand organellar sorting yields chimeric plants consisting of sectors ofwild-type and transgenic cells (Maliga, 1993). In the chimeric tissueantibiotic resistance conferred by aadA or neo is not cell autonomous:transplastomic and wild-type sectors are both green due to phenotypicmasking by the transgenic tissue. Chimerism necessitates a second cycleof plant regeneration on a selective medium. In the absence of a visualmarker this is an inefficient process, involving antibiotic selectionand identification of transplastomic plants by PCR or Southern probing.The feasibility of visual identification of transformed sectors greatlyreduces the effort required to obtain homoplastomic clones.

The Aequorea victoria green fluorescent protein (GFP) is a visualmarker, allowing direct imaging of the fluorescent gene product inliving cells without the need for prolonged and lethal histochemicalstaining procedures. Its chromophore forms autocatalytically in thepresence of oxygen and fluoresces green when absorbing blue or UV light(Prasher et al., 1992; Chalfie et al., 1994; Heim et al., 1994)(reviewed in ref. Prasher, 1995; Cubitt et al., 1995; Misteli andSpector, 1997). The gfp gene was modified for expression in the plantnucleus by removing a cryptic intron, introducing mutations to enhancebrightness and to improve GFP solubility (Pang et al., 1996; Reichel etal., 1996; Rouwendal et al., 1997; Haseloff et al., 1997; Davis andVierstra, 1998). GFP was used to monitor protein targeting to nucleus,cytoplasm and plastids from nuclear genes (Sheen et al., 1995; Chiu etal., 1996; K{hacek over (s)}hler et al., 1997), and to follow virusmovement in plants (Baulcombe et al., 1995; Epel et al., 1996). GFP hasalso been used to detect transient gene expression in plastids (Hibberdet al., 1998).

The expression of GFP by directly incorporating the gfp gene in theplastid genome is described herein. Incorporation of a visual marker,the GFP protein, in the plastid transformation vectors of the presentinvention facilitates distinction of spontaneous antibiotic resistantmutants and plastid transformants (Svab et al., 1990). Furthermore,transplastomic sectors in the chimeric tissue can be visuallyidentified, significantly reducing the time and effort required forobtaining genetically stable transplastomic lines. The utility of theGFP marker described here is further enhanced by its fusion with theenzyme aminoglycoside 3″-adenylyltransferase [AAD] conferringspectinomycin and streptomycin resistance to plants. Using a marker geneencoding a bifunctional protein, FLARE-S (fluorescent antibioticresistance enzyme, spectinomycin and streptomycin), prevents physicalseparation of the two genes and simplifies engineering. Furthermore,fluorescent antibiotic resistance genes enables extension of plastidtransformation to cereal crops, in which plastid transformation is notassociated with a readily identifiable tissue culture phenotype.

The following protocols are provided to facilitate the practice of thepresent example.

Construction of tobacco plastid vectors. The aadA16gfp gene encodesFLARE16-S fusion protein, and can be excised as an NheI-XbaI fragmentfrom plasmid pMSK51, a pBSKSII+ derivative (Genbank Accesssion No. Notyet assigned. The fusion protein was obtained by cloning gfp (fromplasmid pCD3-326F) downstream of aadA (in plasmid pMSK38), digesting theresulting plasmid with BstXI (at the 3′ end of the aadA coding region)and NcoI (including the gfp translation initiation codon) and linkingthe two coding regions by a BstXI-NcoI compatible adapter. The adapterwas obtained by annealing oligonucleotides5′-GTGGGCAAAGAACTTGTTGAAGGAAAATTGGAGCTAGTAGAAGGTCTTAAAGT CGC-3′ (SEQ IDNO: 88) and 5′-CATGGCGACTTTAAGACCTTCTACTAGCTCCAATTTTCCTTCAACAAGTTCTTTGCCCACTACC-3′ (SEQ ID NO: 89). The adapter connects AAD and GFP with apeptide of 16 amino acid residues (ELVEGKLELVEGLKVA; SEQ ID NO: 104).

The engineered aadA gene (Chinault et al., 1986) in plasmid pMSK38(pBSIIKS+ derivative) has NcoI and NheI sites at the 5′ end and BstXIand XbaI sites at the 3′ end of the gene. The NcoI site includes thetranslation initiation codon; the NheI and BstXI sites are in the codingregion close to the 5′ and 3′ ends, respectively; the XbaI site isdownstream of stop codon. The mutations were introduced by PCR usingoligonucleotides 5′-GGCCATGGGGGCTAGCGAAGCGGTGATCGCCGAAGTATCG-3′ (SEQ IDNO: 90) and 5′-CGAATTCTAGACATTATTTGCCCACTACCTTGGTGATCTC-3′ (SEQ ID NO:91).

The gfp gene in plasmid CD3-326F is the derivative of plasmid psmGFP,encoding the soluble modified version of GFP (accession number U70495)obtained under order number CD3-326 from the Arabidopsis BiologicalResource Center, Columbus, Ohio (Davis and Vierstra, 1998). The gfp genein plasmid CD3-326F is expressed in the PpsbA/TpsbA expression cassette.The gfp gene in plasmid CD3-326F was obtained through the followingsteps. The BamHI-SacI fragment from CD3-326 was cloned into pBSKS+vector to yield plasmid CD3-326A. The SacI site downstream of the codingregion was converted into an XbaI site by blunting and linker ligation(5′-GCTCTAGAGC; SEQ ID NO: 107; plasmid CD3-326B). An NcoI site wascreated to include the translation initiation codon and at the same timethe internal NcoI site was removed by PCR amplification of the codingregion N-terminus with primers5′-CCGGATCCAAGGAGATATAACACCATGGCTAGTAAAGGAGAAGAACTTTTC-3′ (SEQ ID NO:92) and 5′-GTGTTGGCCAAGGAACAGGTAGTTTTCC-3′ (SEQ ID NO: 93). ThePCR-amplified fragment was digested with BamHI and MscI restrictionenzymes, and the resulting fragment was used to replace the BamHI-MscIfragment in plasmid CD3-326B to yield plasmid CD3-326C. The gfp codingregion was excised from plasmid CD3-326C as an NcoI-XbaI fragment andcloned into a psbA cassette to yield plasmid CD3-326D. PpsbA and TpsbAare the psbA gene promoter and 3′-untranslated region derived fromplasmids pJS25 (Staub and Maliga, 1993). TpsbA has been truncated byinserting a HindIII linker downstream of the modified BspHI site (PeterHajdukiewcz, unpublished). The PpsbA::gfp::TpsbA gene was excised as anEcoRI-HindIII fragment and cloned into EcoRI and HindIII digestedpPRV111A, to yield plasmid CD3-326F.

The chimeric aadA16gfp genes were introduced into the tobacco plastidtransformation vector pPRV111B (Zoubenko et al., 1994). The aadA genewas excised from plasmid pPRV111B with EcoRI and SpeI restrictionenzymes, and replaced with the EcoRI-SpeI fragment from plasmids pMSK53and pMSK54 to generate plasmids pMSK57 (aadA16gfp-S2) and pMSK56(aadA16gfp-S1).

Construction of rice plastid vectors. Plasmid pMSK49 is a rice-specificplastid transformation vector which carries the aadA11gfp-S3 gene as theselective marker in the trnV/rps12/7 intergenic region (GenBankAccession Number: Not yet assigned). Plasmid pMSK49 carries the riceSmaI-SnaBI plastid fragment (restriction sites at nucleotides 122488 and125 878 in the genome Hiratsuka et al., 1989) cloned into a pBSKSII+(Stratagene) vector after blunting the SacI and KpnI restriction sites.The XbaI site present in the rice plastid DNA fragment (position atnucleotide 125032 in the genome (Hiratsuka et al., 1989) was removed byfilling in and religation. Prior to cloning the selective marker theprogenitor plasmid was digested with the BglII restriction enzyme givingrise to a deletion of 119 nucleotides between two proximal BglII sites(positions at 124367 and 124491). The aadA11gfp-S3 gene was then clonedin the blunted BglII sites.

The aadA gene in plasmid pMSK49 was obtained by modifying the aadA genein plasmid pMSK38 (above) to obtain plasmid pMSK39. The modificationinvolved translationally fusing the aadA gene product at its N-terminuswith an epitope of the human c-Myc protein (amino acids 410-419;EQKLISEEDL; SEQ ID NO: 106; Kolodziej and Young, 1991). The geneticengineering was performed by ligating an adapter obtained by annealingcomplementary oligonucleotides with appropriate overhangs into NcoI-NheIdigested pMSK38 plasmid. The oligonucleotides were:5′-CATGGGGGCTAGCGAACAAAAA CTCATTTCTGAAGAAGACTTGc-3′ (SEQ ID NO: 94) and5′-CTAGGCAAGTCTTCTTCAGAAATGAGTTTTTGTTCGCTAGCCCC-3′ (SEQ ID NO: 95).

The aadA11gfp gene encoding FLARE11-S was obtained by linking AAD andGFP with the 11-mer peptide ELAVEGKLEVA (SEQ ID NO: 105). To clone aadAand gfp in the same polycloning site, gfp (EcoRI-HindIII fragment; fromplasmid CD3-326F) was cloned downstream of aadA in plasmid pMSK39 toobtain plasmid pMSK41. The two genes were excised together as anNheI-HindIII fragment, and cloned into plasmid pMSK45 to replace akanamycin-resistance gene yielding plasmid pMSK48. Plasmid pMSK45 is aderivative of plasmid pMSK35 which carries the PrrnLT7g10+DB/Ecpromoter. The promoter consists of the plastid rRNA operon promoter andthe leader sequence of the T7 phage gene 10 leader. In plasmid pMSK48,aadA is expressed from the PrrnLT7g10+DB/Ec promoter. The aadA and gfpgenes were then translationally fused with an BstXI-NcoI adapter thatlinks the AAD and GFP with an 11-mer peptide. The adapter was obtainedby annealing oligonucleotides5′-GTGGGCAAAGAACTTGCAGTTGAAGGAAAATTGGAGGTCGC-3′ (SEQ ID NO: 96) and5′-CATGGCGACCTCCAATTTTCCTTCAACTGCAAGTTCTTTGCCCACTACC-3′ (SEQ ID NO: 97),which was ligated into BstXI/NcoI digested pMSK48 plasmid DNA to yieldplasmid pMSK49. Plasmid pMSK49 has the rice plastid targeting sequencespresent in plasmid pMSK35.

Tobacco plastid transformation. Tobacco leaves from 4 to 6 weeks oldplants were bombarded with DNA-coated tungsten particles using theDupont PDS1000He Biolistic gun (1100 psi). Transplastomic clones wereidentified as green shoots regenerating on bleached leaf sections onRMOP medium containing 500 mg/L spectinomycin dihydrochloride (Svab abdMaliga, 1993). The spectinomycin resistant shoots were illuminated withUV light (Model B 100AP, UV Products, Upland, Calif., USA). Shootsemitting green light were transferred to spectinomycin free MS medium(Murashige and Skoog, 1962) (3% sucrose) on which fluorescent(transplastomic) and non-fluorescent (wild-type) sectors formed.Fluorescent sectors were excised, and transferred to selective (500 mg/Lspectinomycin) shoot regeneration (RMOP) medium. Regenerated shoots weretested for uniform transformation by Southern analysis.

Rice plastid transformation. Callus formation from mature Oryza sativacv. Taipei 309 seeds was induced on a modified CIM medium (Tompson etal., 1986), containing MS salts and vitamins (2 mg/L glycine, 0.5 mg/Lnicotinic acid, 0.5 mg/L pyridoxine and 0.1 mg/L thiamine), 2 mg/L 2,4D,1 mg/L kinetin and 300 mg/L casein enzymatic hydrolysate Type III (SigmaC-1026) and sucrose (30 g/L). Embryogenic suspensions from theproliferating embryogenic calli were obtained on the AA medium (Mullerand Grafe, 1978). For plastid transformation by the biolistic processrice embryogenic cells were plated on a filter paper on non-selectivemodified CIM medium (Tompson et al., 1986). The bombarded cells wereincubated for 48 hours, transferred to selective liquid AA medium(Muller and Grafe, 1978) (one to two weeks), and then to solid modifiedRRM regeneration medium (Zhang and Wu, 1988) containing MS salts andvitamins, 100 mg/L myo-inositol, 4 mg/L BAP, 0.5 mg/L IAA, 0.5 mg/L NAA,30 g/L sucrose and 40 g/L maltose and 100 mg/L streptomycin sulfate onwhich green shoots appeared in two to three weeks. The shoots wererooted on a selective MS salt medium (Murashige and Skoog, 1962)containing 30 g/L sucrose and 100 mg/L streptomycin sulfate. Leafsamples for PCR analysis and confocal microscopy were taken from plantson selective medium.

PCR amplification of border fragments. Total cellular DNA was extractedaccording to Mettler (Mettler, 1987). The PCR analysis was carried outwith a 9:1 mixture of AmpliTaq (Stratagene) and Vent (New EnglandBiolabs) DNA polymerases in the Vent buffer following the manufacturer'srecommendations. The left border fragment was amplified with primers O3(5′-ATGGATGAACTATACAAATAAG-3′; SEQ ID NO: 98) and O4(5′-GCTCCTATAGTGTGACG-3′; SEQ ID NO: 99). The right border fragment wasamplified with primers O5 (5′-ACTACCTCTGATAGTTGAGTCG-3′; SEQ ID NO: 100)and O6 (5′-AGAGGTTAATCGTACTCTGG-3′; SEQ ID NO: 101). The aadA part ofFLARE-S genes was amplified with primers O1(5′-GGCTCCGCAGTGGATGGCGGCCTG-3′; SEQ ID NO: 102) and O2(5′-GGGCTGATACTGGGCCGGCAGG-3′; SEQ ID NO: 103). Primer positions areshown in FIG. 5A. Note that the same primers can be used intransplastomic tobacco and rice plants expressing FLARE-S.

Detection of FLARE-S by fluorescence. FLARE-S expressing sectors in theleaves were visualized by an Olympus SZX stereo microscope equipped forGFP detection with a CCD camera system. Subcellular localization of GFPwas verified by laser-scanning confocal microscopy (Sarastro 2000Confocal Image System, Molecular Dynamics, Sunnyvale, Calif.). Thissystem includes an argon mixed gas laser with lines at 488 and 568 nmand detector channels. The channels are adjusted for fluorescein andrhodamine images. GFP fluorescence was detected in the FITC channel(488-514 nm). Chlorophyll fluorescence was detected in the TRITC channel(560-580 nm). The images produced by GFP and chlorophyll fluorescencewere viewed on a computer screen attached to the microscope andprocessed using the Adobe PhotoShop software.

Immunoblot analysis. Leaves (0.5 g) collected from plants in sterileculture were frozen in liquid nitrogen and ground to a fine powder in amortar with a pestle. For protein extraction the powder was transferredto a centrifuge tube containing 1 ml buffer [50 mM Hepes/KOH (pH 7.5), 1mM EDTA, 10 mM potassium acetate, 5 mM magnesium acetate, 1 mMdithiothreitol and 2 mM PMSF] and mixed by flicking. The insolublematerial was removed by centrifugation at 4° C. for 5 min at 11,600 g.Protein concentration in the supernatant was determined using the Bioradprotein assay reagent kit. Proteins (20 μl per lane) were separated in12% SDS-PAGE (Laemmli, 1970). Proteins separated by SDS-PAGE weretransferred to a Protran nitrocellulose membrane (Schleicher andSchuell) using a semi-dry electroblotting apparatus (Bio-Rad). Themembrane was incubated with Living Colors Peptide Antibody (Clontech)diluted 1 to 200. FLARE-S was visualized using ECL chemilluminescenceimmunoblot detection on X-ray film. FLARE-S on the blots was quantifiedby comparison with a dilution series of commercially available purifiedwild-type GFP (Clontech).

Results and Discussion

Tobacco Plastid Vectors with FLARE-S as the Selectable Marker.

Two FLARE-S fusion proteins were tested in E. coli. In one, the AAD andGFP were linked by an 11-mer (ELAVEGKLEVA; SEQ ID NO: 105), in thesecond by a 16-mer (ELVEGKLELVEGLKVA; SEQ ID NO: 104) linker. Fortransformation in tobacco, the aadA16gfp coding region (16-mer linker)was expressed in two cassettes known to mediate high levels of proteinaccumulation in plastids. Both utilize the strongest known plastidpromoter driving the expression of the ribosomal RNA operon (Prrn), andthe 3′-UTR of the highly expressed psbA gene (TpsbA) for thestabilization of the chimeric mRNAs. The PrrnLatpB+wtDB (plasmid pMSK56)and PrrnLrbcL+DBwt (plasmid pMSK57) promoters utilize the atpB or rbcLgene leader sequences and the coding region N-termini with thedownstream box (DB) sequence, respectively. Due to inclusion of the DBsequence in the chimeric genes, the proteins encoded by the two genesare slightly different, having 14 amino acids of the ATP-ase β subunit(atpB gene products) or ribulose 1,5-bisphosphate carboxylase/oxygenase(rbcL gene product) translationally fused with FLARE16-S (FLARE16-S1 andFLARE16-S2, respectively). To obtain a plastid transformation vectorwith the fluorescent spectinomycin resistance genes, the chimeric geneswere cloned into the trnV/rps12/7 plastid intergenic region in plastidvector pPRV111B. Plasmids pMSK56 and pMSK57 (FIG. 23) express FLARE16-S1and FLARE16-S2, respectively, as markers.

Identification of transplastomic tobacco clones by fluorescence.Transformation was carried out by biolistic delivery of pMSK56 andpMSK57 plasmid DNA into chloroplast. The bombarded leaves weretransferred onto selective (500 mg/L spectinomycin) shoot regenerationmedium. Wild-type leaves on this medium bleach and form white callus.Cells with transformed plastids regenerate green shoots. The leaves onthe selective medium were regularly inspected with a hand-held long-waveUV lamp for FLARE-S fluorescence.

No fluorescence could be detected in young shoots (3 to 5 mm in size)developing on pMSK56-bombarded leaves. However, formation of brightsectors in the leaves was observed, when these small shoots weretransferred onto non-selective plant maintenance medium. In contrast,cultures bombarded with plasmid pMSK57 yielded small fluorescent shootsat an early stage. These fluorescent shoots, and some of thenon-fluorescent ones, developed into plants with bright sectors onnon-selective plant maintenance medium. Therefore, FLARE16-S2 is usefulfor early detection of plastid transformation events. FLARE16-S2fluorescence in young shoots on a selective medium should be due torelatively high levels of FLARE16-S2. Higher levels of FLARE16-S2 arealso indicated by the brighter sectors in variegated leaves expressingFLARE16-S2 as compared to FLARE16-S1.

The size of sectors was different in individual shoots. FLARE-Sexpression in different leaf layers was also obvious. With thetraditional selection for spectinomycin resistance, the transplastomicand wild-type sectors are not visible. Regeneration of plants withuniformly transformed plastid genomes was greatly facilitated by thefluorescing sectors expressing FLARE-S, which could be readilyidentified in UV light, dissected, and transferred for a second cycle ofplant regeneration on spectinomycin-containing (500 mg/L) selectivemedium.

Given the high levels of FLARE-S accumulation we were interested to findout, if FLARE-S is toxic to plants. We expected that toxicity should bemanifested as lower transformation efficiencies. Bombardment of 30tobacco leaves with plasmids pMSK56 and pMSK57 yielded 71 and 89spectinomycin resistant clones, respectively. Out of these, 61 and 77lines were verified as transplastomic by fluorescence. Plastidtransformation in a subset of these was confirmed by confocal laserscanning microscopy (7 clones each; see below) and Southern analysis (4clones). The frequency of plastid transformation events with theFLARE-S-expressing genes was slightly higher (˜2 instead of ˜1 perbombardment) than reported earlier with a chimeric aadA gene at the sameinsertion site (Svab and Maliga, 1993). Therefore, we assume thataccumulation of FLARE-S at high levels is not detrimental. Lack oftoxicity is also supported by the apparently normal phenotype of theplants in the greenhouse (not shown).

Localization of FLARE-S to tobacco plastids by confocal microscopy. Dueto phenotypic masking, transplastomic and wild type sectors in achimeric leaf are both green on a selective medium. However, we havefound that in chimeric leaf sectors in the same cell some plastidsexpress FLARE-S while others do not, when observed by confocalmicroscopy (FIG. 24). FLARE-S and chlorophyll fluorescence were detectedseparately in the fluorescein and rhodamine channels, respectively. Thetwo images were then overlaid confirming that FLARE-S fluorescencederives from chloroplasts.

Expression of FLARE-S was also studied in non-green plastid typesincluding the chromoplasts in petals and the non-green plastids in rootcells (FIGS. 24 b,f). These studies were carried out in plants, whichwere homoplastomic for the transgenomes. Homoplastomic state wasimportant, since in non-green tissues chlorophyll could not be used forconfirmation of the organelles as plastids. Since FLARE-S expressioncould be readily detected in chloroplasts as well as non-green plastids,the plastid rRNA operon promoter is apparently active in all plastidtypes.

FLARE-S accumulation in tobacco leaves. Accumulation of FLARE-S inhomoplastomic leaves was tested using the commercially available GFPantibody, recognizing the GFP portion (239 amino acid residues) ofFLARE16-S (520 amino acids). FLARE16-S1 (532 amino acids) was ˜8%,whereas FLARE16-S2 (532 amino acids) was ˜18% of total soluble leafprotein (FIG. 25). To calculate FLARE16-S concentrations, a GFP dilutionseries was used as a reference, and the values were than increased by2.6 to correct for the larger size of the FLARE16-S1 and -S2 proteins.

Tracking plastid transformation in rice by FLARE-S expression. In rice,plant regeneration is from non-green embryogenic cells. Encouraged byFLARE-S expression in non-green tobacco plastids, we attempted totransform the non-green plastids of embryogenic rice tissue-culturecells. Plastid transformation was carried out using a rice-specificvector expressing FLARE11-S3 and targeting insertion of the aadA11gfp-S3gene in the trnV/rps12/7 intergenic region. The location of theinsertion site and the size of plastid targeting sequences in the ricevector are similar to the tobacco vectors shown in FIG. 23.

Plastid transformation in rice was carried out by bombardment ofembryogenic rice suspension culture cells using gold particles coatedwith plasmid pMSK49 DNA. Rice cells, as most cereals, are naturallyresistant to spectinomycin (Fromm et al., 1987). FLARE-S, however,confers resistance to streptomycin as well (Svab and Maliga, 1993).Therefore, selection for transplastomic lines was carried out onselective streptomycin medium (100 mg/L). Streptomycin at thisconcentration inhibits the growth of embryogenic rice cells. Afterbombardment, the rice cells were first selected in liquid embryogenic AAmedium, then on the solid plant regeneration medium, on which thesurviving resistant cells regenerated green shoots (12 in 25 bombardedplates). These shoots were rooted, and grown into plants. PCRamplification of border fragments in DNA isolated from the leaves ofthese plants confirmed integration of aadA11gfp-S3 sequences in theplastid genome (FIG. 26). The left and right border fragments can not beamplified if the gene is integrated into the nuclear genome, as one ofthe primers (O4 or O6) of the pairs is outside the plastid targetingregions.

FLARE11-S3 expression in the leaves of two of the PCR-positive plantswas tested by confocal laser-scanning microscopy. In rice, as intobacco, the FLARE-S marker confirmed segregation of transplastomic andwild-type plastids (FIG. 27). In rice only a small fraction ofchloroplasts expressed FLARE-S. Since individual cells marked witharrows in FIG. 27 contained a mixed population of wild-type andtransgenic chloroplasts, FLARE-S in these cells could be expressed onlyfrom the plastid genome. Integration of aadA11gfp-S3 into the nucleargenome downstream of plastid-targeting transit peptide would result inuniform expression of FLARE-S in each of the chloroplasts within thecell.

The sequences of the selectable marker genes of the invention areprovided in FIGS. 28-34. FIG. 35 depicts a table describing theselectable marker genes disclosed in the present example.

Direct visual identification of transplastomic sectors requires highlevel expression of FLARE-S in plastids. High GFP expression levels inArabidopsis were toxic, interfering with plant regeneration. Toxicity ofwild-type (insoluble) GFP was linked to GFP accumulation in the nucleusand cytoplasm, and could be eliminated by targeting it to theendoplasmic reticulum (Haseloff et al., 1997). GFP aggregates were alsocytotoxic to E. coli cells (Crameri et al., 1996). To enhancefluorescence intensity and to avoid cytotoxicity, soluble versions ofthe codon-modified GFP were obtained (Davis and Vierstra, 1998). We haveutilized the gene for a soluble-modified GFP described by Davis andVierstra (Davis and Vierstra, 1998) to create variants of FLARE-S, afusion protein, which does not have an apparent cytotoxic effect. Thefrequency of plastid transformation, if affected at all, is increasedrather then decreased. In tobacco, we normally obtain one transplastomicclone per bombarded leaf sample (Svab and Maliga, 1993), whereas withthe FLARE-S genes on average we could recover two clones per sample.Plant regeneration from highly fluorescent tissue was readily obtained,and the regenerated plants have a phenotype indistinguishable from thewild type.

Plastid transformation in rice requires expression of the selectivemarker in non-green plastids. The rRNA operon has two promoters, one forthe eubacterial-type (PEP) and one for the phage-type (NEP) plastid RNApolymerase. The promoter driving FLARE-S expression is recognized onlyby the eubacterial-type plastid RNA polymerase. Previously, it wasassumed that the eubacterial-type promoter is active only inchloroplasts (Maliga, 1998). Accumulation of FLARE-S in roots and petalsindicates that PEP is also active in non-green plastids.

Plastid transformation is a process that unavoidably yields chimericplants, since cells of higher plants contain a large number (300 to50000) of plastid genome copies (Bendich, 1987), out of which initiallyonly a few are transformed. High level expression of FLARE-S in plastidsprovides the means for visual identification of transplastomic sectors,even if they are present in a chimeric tissue. GFP and AAD could beexpressed from two different genes in a plastid transformation vector.However, transformation with a marker gene encoding a bifunctionalprotein prevents separation of the two genes and simplifies engineering.The fluorescent selective marker will significantly reduce the workrequired to obtain genetically stable plastid transformants in tobacco,a species in which plastid transformation is routine. The bottleneck ofapplying plastid transformation in crop improvement is the lack oftechnology. In tobacco, chimeric clones with transformed plastids arereadily identified by shoot regeneration (Svab et al., 1990). InArabidopsis, clones with transformed plastids are identified by greening(Sikdar et al., 1998). We have shown here that FLARE-S is a suitablemarker to select for transplastomes in embryogenic rice cells, whichlack the visually identifiable tissue culture phenotypes exploited intobacco and Arabidopsis. Data presented here are the first example forstable integration of foreign DNA into the rice plastid genome. Theserice plants are heteroplastomic. Uniformly transformed rice plants willbe obtained by further selection on streptomycin medium and screeningthe embryogenic cells for FLARE-S expression. Thus, the FLARE-S markersystem will enable extension of plastid transformation to cereal crops.

The Utility of the New Chimeric Promoters

The σ⁷⁰-type plastid ribosomal RNA operon promoter, Prrn, is thestrongest known plastid promoter expressed in all tissue types. Theultimate product of this promoter in the plastid is RNA not protein.Therefore, a series of chimeric promoters were constructed to facilitateprotein accumulation from Prrn, using expression of the neomycinphosphotransferase (NPTII) enzyme as the reference protein.

1) The expression cassettes have distinct tissue-specific expressionprofiles. Some of the expression cassettes described here willfacilitate relatively high levels of protein expression in all tissues,including leaves, roots and seeds. Other cassettes have differentexpression profiles: for example will facilitate moderate levels ofprotein accumulation in the leaves while lead to relatively high levelsof protein accumulation in the roots. Accumulation of a protein atlevels of 10% to 50% of total soluble protein is considered high-levelprotein expression; low-levels of protein expression would be in therange of ≦0.1% total soluble cellular protein.

2) Efficiency of the selectable marker gene depends on the rate at whichthe gene product accumulates during the early stage of transformation.Since initially present only in a few copies per cell, high levels ofexpression from a few copies will provide protection from toxicsubstances early on, facilitating efficient recovery of transformedlines. The expression cassettes will be useful to drive the expressionof the genes conferring resistance to the antibiotics streptomycin,spectinomycin and hygromycin, and the herbicides phosphinotrycin andglyphosate. In such applications addition of amino acids at theN-terminus is acceptable, as long as it does not interfere with theexpression of the selectable marker genes. NPTII is such an enzyme. Incases like NPTII, an N-terminal fusion and thereby the mRNA “DownstreamBox” sequences give an additional at least two to four-fold increase inprotein levels. The −DB construct which relied on an NheI site, andinvolved addition of one (N-terminal) amino acid of the source genecoding region is convenient, but is not necessary. When translationalfusion is not feasible due to inactivation of proteins, seamlessin-frame constructs may be created by PCR methods outlined in theapplication.

3) A second major area on which application of the chimeric promoters isextremely useful is protein expression for pharmaceutical, industrial oragronomic purposes. The examples include, but are not restricted to,production of vaccines, healthcare products like human hemoglobin,industrial or household enzymes.

REFERENCES

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A recombinant DNA construct for expressing at least one heterologousprotein in the plastids of higher plants, said construct comprising achimeric 5′ regulatory region which includes a promoter element, aleader sequence and a downstream box element operably linked to a codingregion of said at least one heterologous protein, said chimericregulatory region enhancing translational efficiency of an mRNA moleculeencoded by said DNA construct, relative to constructs lacking saidchimeric regulatory region.
 2. A vector comprising the DNA construct ofclaim
 1. 3. The recombinant DNA construct as claimed in claim 1, said 5′regulatory region being selected from the group consisting ofPrnnLatpB+DBwt, SEQ ID NO:1, PrrnLatpB−DB, SEQ ID NO:2, PrrnLatpB+DBm,SEQ ID NO:3, PrrnLclpP+DBwt, SEQ ID NO: 4, PrrnclpP−DB, SEQ ID NO:5,PrrnLrbcL+DBwt, SEQ ID NO:6, PrrnLrbcL−DB, SEQ ID NO:7, PrrnLrbcL+DBm,SEQ ID NO:8, PrrnLpsbB+DBwt, SEQ ID NO:9, PrrnLpsbB−DB, SEQ ID NO:10,PrrnLpsbA+DBwt, SEQ ID NO: 11, PrrnLpsbA−DB, SEQ ID NO:12,PrrnLpsbA−DB(+GC), SEQ ID NO:13.
 4. The recombinant DNA construct asclaimed in claim 1, said chimeric 5′ regulatory region being selectedfrom the group consisting of PrrnLT7g10+DB/pt, SEQ ID NO:15,PrrnLT7g10−DB SEQ ID NO:16.
 5. A vector comprising the DNA construct asclaimed in claim
 4. 6. The DNA construct as claimed in claim 1, saiddownstream box element having a sequence selected from the groupconsisting of 5′TCCAGTCACTAGCCCTGCCTTCGGCA′3 (SEQ ID NO: 29) and5′CCCAGTCATGAATCACAAAGTGGTAA′3 (SEQ ID NO:30).
 7. The DNA construct asclaimed in claim 1, wherein said heterologous protein is expressed froma bar gene encoded by S. hydroscopicus said bar gene being inserted intoa plasmid selected from the group consisting of pKO12, and pJEK3, saidpJEK3 having the sequence of SEQ ID NO:
 18. 8. The DNA construct asclaimed in claim 1, wherein said heterologous protein is expressed froma synthetic bar encoding nucleic acid, wherein said synthetic barencoding nucleic acid is selected from the group consisting of SEQ IDNO: 19 and SEQ ID NO:20.
 9. The DNA construct as claimed in claim 1,said at least one heterologous protein comprising a fusion protein. 10.The DNA construct as claimed in claim 9, said fusion protein having afirst and second coding region operably linked to said chimeric 5′regulatory region such that production of said fusion protein isregulated by said chimeric 5′ regulatory region, said first codingregion encoding a selectable marker gene and said second coding regionencoding a fluorescent molecule to facilitate visualization oftransformed plant cells.
 11. A vector comprising the DNA construct ofclaim
 10. 12. The DNA construct as claimed in claim 9, said fusionprotein being encoded by a polynucleotide consisting of an aadA codingregion operably linked to a green fluorescent protein coding region. 13.The DNA construct as claimed in claim 12, said aadA coding region beingoperably linked to said green fluorescent protein coding region via anucleic acid molecule encoding a peptide linker having comprising anamino acid sequence selected from the group consisting ofELVEGKLELVEGLKVA (SEQ ID NO:104) and ELAVEGKLEVA (SEQ ID NO:105). 14.The DNA construct as claimed in claim 10, said construct comprising asequence selected from the group of SEQ ID NOS: 21-25 21, 22, 23, 24,25, and
 27. 15. A plasmid for transforming the plastids of higherplants, said plasmid being selected from the group consisting ofpHK30(B), pHK31(B), pHK60, pHK32(B), pHK33(B), pHK34(A), pHK35(A),pHK64(A), pHK36(A), pHK37(A), pHK38(A), pHK39(A), pHK40(A), pHK41(A),pHK42(A), pHK43(A), pMSK56, pMSK57, pMSK45, pMSK48, pMSK49, and pMSK35,pMSK53 and pMSK54.
 16. A transgenic plant containing the plasmid asclaimed in claim
 15. 17. The transgenic plant as claimed in claim 15,said plant being selected from the group consisting of monocots anddicots.
 18. A method for producing transplastomic monocots, comprising:a) obtaining embryogenic cells; b) exposing said cells to a heterologousDNA molecule under conditions whereby said DNA enters the plastids ofsaid cells, said heterologous DNA molecule encoding at least oneexogenous protein, said at least one exogenous protein encoding aselectable marker; c) applying a selection agent to said cells tofacilitate sorting of untransformed plastids from transformed plastids,said cells containing transformed plastids surviving and dividing in thepresence of said selection agent; d) transferring said surviving cellsto selective media to promote shoot regeneration and growth; and e)rooting said shoots, thereby producing transplastomic monocot plants.19. The method as claimed in claim 18, wherein said heterologous DNAmolecule is introduced into said plant cell via a process selected fromthe group consisting of biolistic bombardment, Agrobacterium-mediatedtransformation, microinjection and electroporation.
 20. The method asclaimed in claim 18, wherein protoplasts are obtained from saidembryogenic cells and said heterologous DNA molecule is delivered tosaid protoplasts by exposure to polyethylene glycol.
 21. The method asclaimed in claim 18, wherein said selection agent is selected from thegroup consisting of streptomycin, and paromomycin
 22. A monocottransformed via the method of claim
 18. 23. A transformed monocot plantas claimed in claim 22, said monocot plant being selected from the groupconsisting of maize, millet, sorghum, sugar cane, rice, wheat, barley,oat, rye, and turf grass.
 24. A method for producing transplastomic riceplants, said method comprising: a) obtaining embryogenic calli; b)inducing proliferation of calli on modified CIM medium; c) obtainingembryogenic cell suspensions of said proliferating calli in liquid AAmedium; d) bombarding said embryogenic cells with microprojectilescoated with plasmid DNA; e) tranferring said bombarded cells toselective liquid AA medium; f) transferring said cells surviving in AAmedium to selective RRM regeneration medium for a time period sufficientfor green shoots to appear; and g) rooting said shoots in a selective MSsalt medium.
 25. The method as claimed in claim 24, said plasmid DNAbeing selected from the group of plasmids consisting of pMSK35 andpMSK53, pMSK54 and pMSK49.
 26. A transplastomic rice plant produced bythe method of claim
 24. 27. A method for containing transgenes intransformed plants, comprising: a) determining the codon usage in saidplant to be transformed and in microbes found in association with saidplant; and b) genetically engineering said transgene sequence via theintroduction of rare codons to abrogate expression of said transgene insaid plant associated microbe.
 28. A method as claimed in claim 27,wherein said transgene is a bar gene and said rare codons are arginineencoding codons selected from the group consisting of AGA and AGG.